Objective To investigate the effects of heat preconditioning (HP) on the expression of protein kinase C (PKC) δ and PKCε in the myocardium of overload exercise rats. Methods 25 male three-month-old Sprague-Dauley rats were divided into control group (n=5), exercise group (n=10) and HP group (n=10). The expression of PKCδ and PKCε in the myocardium was detected with immunohistochemistry, real-time PCR and Western blotting 8 weeks after overload exercise. Results The expression of PKCδ significantly increased in the exercise group compared with the control group and the HP group (P<0.05). In another hand, PCR and immunohistochemistry showed the expression of PKCε in the exercise group decreased compared with the control group and the HP group (P<0.05), but it was not significantly different among the groups using Western blotting (P>0.05). Conclusion PKCs may play an important role in the HP-induced cardio-protection during overload exercise.