Aim To observe the cleavage of nucleolin (also named C23) during apoptosis induced by oxidative stress and clarify the effect of heat shock protein 70 (HSP70) on cleavage of nucleolin and its possible molecular mechanism. Methods 0.5 mmol/L hydrogen peroxide (H 2O 2) was added into cultured cells to mimic oxidative stress. Cleavage of C23 were detected by using immunoblotting; Heat shock response (HSR) and HSP70 transgenic cell lines were used to observe the effect of HSR and HSP70 on cleavage of C23 induced by oxidative stress, and the relationship between HSP70 and C23 was evaluated by using co-immunoprecipitation. Results Activity of caspase-3 increased significantly after 2 h of 0.5 mmol/L H 2O 2 treatment, and reached peak at 12 h. The cleavage of C23 appeared 30 min to 1 h after treatment of H 2O 2 as indicated by a cleaved fragmentation of 80 kDa, which was significantly inhibited by HSR and HSP70. Furthermore co-immunoprecipitation study indicated that HSP70 could bind directly to C23 during H 2O 2 treatment. Conclusions Oxidative stress could induce the activation of caspase-3, cleavage of C23 and apoptosis; and HSP70 could inhibit significantly the cleavage of C23 induced by oxidative stress and its mechanism was related to the interaction between HSP70 and C23 during oxidative stress.