OBJECTIVES: Resistance training combined with consumption of a high-protein diet (HPD) is typically recommended to increase muscle mass, as both acute resistance exercise (RE) and dietary protein intake stimulate mechanistic target of rapamycin complex 1 (mTORC1) and muscle protein synthesis (MPS). However, the effect of chronic HPD consumption on MPS response to an acute RE remains to be determined. METHODS: Male Sprague-Dawley rats aged 10 wk were fed HPD (50 kcal % protein, for 4 wk) or normal protein diet (NPD; 20 kcal % protein). After the 4-wk dietary intervention, the rats were fasted overnight and the right gastrocnemius muscle was subjected to percutaneous electrical stimulation to mimic acute RE, whereas the left gastrocnemius muscle served as control. The rats were sacrificed 6 h after exercise and the tissues were sampled immediately. RESULTS: The HPD group showed significantly lower fat mass and higher skeletal muscle mass than the NPD group without affecting body weight. Resting mTORC1 activity did not differ between the groups. Additionally, resting MPS was also unchanged after HPD. Acute RE significantly increased mTORC1 activity and MPS in both groups. However, differences in diet did not influence the response of mTORC1 activation to acute RE. Furthermore, HPD did not affect the response of MPS to acute RE. CONCLUSION: The present results suggested that although 4 wk of HPD reduces body fat and increases skeletal muscle mass, it does not affect muscle protein synthesis at basal state, and in response to acute RE.