Objective To compare the routine semen parameters，sperm morphology，sperm mitochondrial membrane potential（MMP），and sperm DNA fragmentation index（DFI） of frozen semen specimens in the high-fertility，medium-fertility，and low-fertility groups from a human sperm bank used for artificial insemination with donor semen（AID），and to investigate the association of the above indicators with AID outcome to select useful indicators for assessing the fertility of sperm donors. Methods We collected 140 frozen semen specimens of the same batch used for AID from a reproductive center，including 44 specimens in the low-fertility group，48 specimens in the medium-fertility group，and 48 specimens in the high-fertility group. Sperm MMP and DFI were measured by flow cytometry. Routine semen parameters，the percentage of morphologically normal sperm，sperm MMP，and sperm DFI were compared between the groups. Results There were no significant differences in routine semen parameters and the percentage of morphologically normal sperm between the groups（P>0.05）. Sperm MMP and DFI were significantly different between the groups（P<0.01）. The clinical pregnancy rate of AID showed a significant negative correlation with sperm DFI（r=-0.313，P<0.01）. Conclusion Sperm MMP and sperm DFI are important indicators for sperm function evaluation. Further studies are needed to determine whether sperm MMP can be a completely independent indicator of sperm quality and whether sperm MMP combined with DFI can predict pregnancy outcome in AID.