Levofloxacin has been reported to have cytotoxicity to chondrocytes in vitro. And 17β-estradiol has been widely studied for its protective effects against cell apoptosis. Based on apoptotic cell model induced by levofloxacin, the purpose of this study was to explore the mechanism by which 17β-estradiol protects rat nucleus pulposus cells from apoptosis. Inverted phase-contrast microscopy, flow cytometry, and caspase-3 activity assay were used to find that levofloxacin induced marked apoptosis, which was abolished by 17β-estradiol. Interestingly, estrogen receptor antagonist, ICI182780, and functional blocking antibody to α2β1 integrin, both prohibited the effect of 17β-estradiol. Simultaneously, levofloxacin decreased cellular binding ability to type II collagen, which was also reversed by 17β-estradiol. Furthermore, western blot and real-time quantitative PCR were used to find that integrin α2β1 was responsible for estrogen-dependent anti-apoptosis, which was time-response and dose-response effect. 17β-estradiol was proved for the first time to protect rat nucleus pulposus cells against levofloxacin-induced apoptosis by upregulating integrin α2β1 signal pathway.