Triple negative breast cancer (TNBC) is a highly malignant tumor with a poor prognosis, and the mutation frequency of p53 gene in TNBC patients is more than 80%. Gamabufotalin (CS-6), a skin secretion from Japanese common toad (Bufo japonicus), has been shown to have anticancer effects. However, the role and underlying mechanism of CS-6 against TNBC have not been reported. Herein, the effects of different concentrations of CS-6 on the proliferation of TNBC cell lines MDA-MB-468 and MDA-MB-231 were de-tected by MTT assay and clone formation assay. The effects of CS-6 on the expression of p53 and heat shock protein 90 (HSP90) at exogenous and endogenous levels were detected by Western-blot. The synergistic effect of CS-6 and the HSP90 inhibitor tanespimycin (17-AAG) on the proliferation of MDA-MB-468 and MDA-MB-231 cells was investigated by combination index and equivalent linearization analysis. The results showed that CS-6 significantly inhibited the proliferation of MDA-MB-468 and MDA-MB-231 cells. The mechanism of inhibition of the tumor cell proliferation may be related to degradation of HSP90/mutant p53 protein. In addition, CS-6 and HSP90 inhibitor 17-AAG had a synergistic inhibitory effect on the prolifera-tion of MDA-MB-468 and MDA-MB-231 cells.