Objective To discuss the effects of leucovorin on tumor growth and angiogenesis in the mice with lung cancer transplantation tumor， and to elucidate its mechanism. Methods Thirty-two healthy C57BL/6 mice were selected to prepare the Lewis lung cancer transplantation tumor models. The mice were randomly divided into model group （0.9% NaCl）， low dose of leucovrin group （25 mg·kg^－1 leucovrin）， high dose of leucovrin group （50 mg·kg^－1 leucovrin） and positive control group （60 mg·kg^－1 cyclophosphamide）， and there were 8 mice in each group. The levels of interleukin-2 （IL-2）， interferon-γ （INF-γ） and tumor necrosis factor-α （TNF-α） in serum of the mice in various groups were detected by enzyme linked immunosorbent assay（ELISA） method； the expression of vascular endothelial growth factor （VEGF） in tumor tissue of the mice in various groups were detected by immunohistochemistry， and the microvascular density （MVD） and VEGF protein expression scores were calculated； the expression levels of nuclear factor-κB（NF-κB） and hypoxia inducible factor-1α（HIF-1α） in tumor tissue of the mice in various groups were detected by Western blotting method. Results Compared with model group， the transplantation tumor masses of the mice in low and high doses of leucovorin groups and positive control group were decreased （P<0.05）； compared with low dose of leucovorin group， the transplantation tumor masses of the mice in high dose of leucovorin group and positive control group were decreased （P<0.05）， and the tumor inhibitory rates were increased （P<0.05）； compared with high dose of leucovorin group， the transplantation tumor mass of the mice in positive control group was decreased （P<0.05）， and the tumor inhibitory rate was increased （P<0.05）. Compared with model group， the spleen indexes and thymus indexes of the mice in low and high doses of leucovorin groups and positive control group were increased （P<0.05）； compared with low dose of leucovorin group， the spleen indexes and thymus indexes of the mice in high dose of leucovorin group and positive control group were increased （P<0.05）； compared with high dose of leucovorin group， the spleen index and thymus index of the mice in positive control group were increased （P<0.05）.The ELISA method results showed that compared with model group， the levels of IL-2， INF-γ and TNF-α in serum of the mice in low and high doses of leucovorin groups and positive control group were increased （P<0.05）； compared with low dose of leucovorin group， the levels of IL-2， INF-γ and TNF-α in serum of the mice in high dose of leucovorin group and positive control group were increased （P<0.05）； compared with high dose of leucovorin group， the levels of IL-2， INF-γ and TNF-α in serum of the mice in positive control group were increased（P<0.05）. Compared with model group， the MVD in tumor tissue of the mice in low and high dose of leucovorin groups and positive control group was decreased （P<0.05）； compared with low dose of leucovorin group， the MVD in tumor tissue of the mice in high dose of leucovorin group and positive control group was decreased （P<0.05）； compared with high dose of leucovorin group， the MVD in tumor tissue of the mice in positive control group was decreased （P<0.05）. Compared with model group， the expression amounts of VEGF protein in tumor tissue of the mice in low and high dose of leucovorin groups and positive control group were decreased； compared with low dose of leucovorin group， the expression amounts of VEGF protein in tumor tissue of the mice in high dose of leucovorin group and positive control group were decreased； compared with high dose of leucovorin group， the expression amount of VEGF protein in tumor tissue of the mice in positive control group was decreased. The differences in VEGF protein expression scores between model group， low dose of leucovorin group， high dose of leucovorin group and positive control group were statistically significant （P<0.05）. Compared with model group， the expression levels of NF-κB and HIF-1α in tumor tissue of the mice in low and high doses of leucovorin groups and positive control group were decreased（P<0.05）； compared with low dose of leucovorin group， the expression levels of NF-κB and HIF-1α in tumor tissue of the mice in high dose of leucovorin group and positive control group were decreased （P<0.05）； compared with high dose of leucovorin group， the expression levels of NF-κB and HIF-1α in tumor tissue of the mice in positive control group were decreased （P<0.05）. Conclusion Leucolorin can inhibit the tumor growth， protect the immune organs and suppress the tumor angiogenesis in the Lewis lung cancer transplantation tumor mice， and may exert the therapeutic effect by targeting NF-κB/HIF-1α signaling pathway and down-regulating the expression levels of NF-κB and HIF-1α proteins.