Objective The aim of the current study is to explore the role of AMPK, a key regulator of cellular energy metabolism, in vascular smooth muscle cell (VSMC) migration in response to physiological cyclic stretch. Methods The Flexcell-5000T mechanical loading system was applied with a physiological cyclic stretch of 10% amplitude and 1.25 Hz frequency to primary rat VSMCs, to simulate the mechanical stimulation of VSMCs in vivo; The protein expression of p-AMPK in VSMCs was detected by Western blotting; VSMC migration was detected by wound healing test. Results Compared with the static group, physiological cyclic stretch loading for 24 h significantly decreased the area of wound healing, suggesting that physiological cyclic stretch inhibited VSMC migration. The protein expression of p-AMPK in VSMCs was increased significantly after physiological cyclic stretch loading for 3 h, and was decreased significantly after 24 h. Under physiological cyclic stretch loading conditions, incubating AMPK inhibitor can significantly reduce the protein expression of p-AMPK after 3 h, and promote VSMC migration after 24 h; incubating AMPK activator AICAR under static conditions significantly increased the protein expression of p-AMPK after 3 h, and weakened VSMC migration after 24 h. Conclusions Physiological cyclic stretch inhibits VSMC migration by increasing the protein expression of p-AMPK, suggesting that the VSMC migration regulated by physiological cyclic stretch is of great significance for maintaining vascular homeostasis.