The hydrogen permeation resistance of Ti-6Al-4V alloy treated by laser peening (LP) was investigated. Electrochemical hydrogen charging, hydrogen permeation experiments were carried out respectively. The residual stress, micro-hardness of hydrogenated and non-hydrogenated specimens were analyzed. The apparent hydrogen diffusion coefficient, lattice hydrogen concentration and hydrogen trap density were compared. LP process and hydrogen diffusion process were numerically simulated by ABAQUS software. The results show that LP can effectively increase the compressive residual stress (CRS) and micro-hardness on the surface of Ti-6Al-4V alloy. LP also promoted the closure of the original micro-cracks by forming a dense strengthening layer on the surface of the material, which reduced the hydrogen concentration and the diffusion coefficient in the crystal lattice. The laser power was inversely related to the hydrogen diffusion coefficient. The numerical simulation results reveal that the hydrogen concentration in the depth direction of LPed specimen decreases significantly, and the maximum drop of 50% for hydrogen concentration was appeared at the depth of 0.6 mm from the top surface. Moreover, the distribution trend of hydrogen concentration in the LP area is consistent with that of CRS, which indicates that CRS plays a positive role in the inhibition of hydrogen diffusion.