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In order to investigate the hydrogen permeation behavior of 316L stainless steel during the microstructural evolution induced by laser peening (LP), an electrochemical hydrogen charging system for initial hydrogen charging of LPed and non-LPed specimen was developed. Afterward, the microhardness, residual stress, and microstructures of the samples were determined and analyzed. Finally, electrochemical hydrogen permeation experiments were undertaken to verify LP's influence on hydrogen permeation parameters of 316L. The results showed that LP reduced the hydrogen-induced hardening rate of the alloy and additionally invoked high magnitude compressive residual stress on its surface. At the layer close to the face of the specimen, the grain refinement rate was as high as 56.18%, which was accompanied by the appearance of high-density dislocations. Compared with the non-LPed sample, the hydrogen permeation time increased significantly, and the saturation current density in steady state hydrogen permeation also decreased gradually.