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The effect of pre-strain before hydrogen charging on the resistance to hydrogen embrittlement (HE) in the 316L austenitic stainless steel was investigated through the slow strain rate tensile test (SSRT), transmission electron microscopy, and thermal desorption analysis (TDA). The pre-strain suppressed mechanical twinning during the SSRT, regardless of hydrogen charging. However, it accelerated the ¾-martensitic transformation in hydrogen-charged specimens. The TDA revealed that whereas hydrogen atoms migrated from grain boundaries and dislocations mainly to the austenite (£)/¾ interfaces in pre-strained specimens during the SSRTs, they moved to the boundaries of fresh mechanical twins, which newly formed during the SSRTs, in the annealed specimen. The elongation loss by hydrogen charging became greater with increasing the pre-strain, indicating that pre-straining deteriorated the resistance to HE. This elongation loss by pre-strain resulted from both the increase in fraction of ¾-martensite with pre-strain and the segregation of hydrogen atoms to the £/¾ interfaces.