Aim To observe the effect of Krppel-like factor 4 (KLF4) overexpression on heat stress-induced apoptosis of C2C12 cells. Methods C2C12 cells transfected with pcDNA3.1 or pcDNA3.1-KLF4 plasmids were exposed to heat stress (42.5℃) for 1 h and recovered at 37℃ for 12 h. Flow cytometry (FCM) and Hoechst33258 staining assays were performed to assess the apoptosis; the expression of Bcl-2 and Bax was detected by Western blots. Results After heat stress, FCM showed that apoptotic cells significantly increased and the percent of apoptosis was 19.6% in the cells overexpressed with KLF4. It was determined were characterized with classical morphologic changes including apoptotic body and nuclear condensation in the cells overexpressed with KLF4.And the expression of Bcl-2 and Bax also significantly changed in the cells overexpressed with KLF4. Conclusion KLF4 overexpression promoted the apoptosis of C2C12 cells.