Abstract: To investigate the effect of twinning crystal fraction on the hydrogen embrittlement behavior of high-manganese steel fabricated by wire and arc additive manufacturing (WAAM), tensile pre-straining was employed to prepare microstructures with different twinning crystal fractions, followed by tests on mechanical properties and subsurface hydrogen concentration. The results show that as the pre-strain level increases from 0% to 5% and 25%, the corresponding twinning crystal fraction rises from 0% to 5.87% and 63.4%, respectively. With the increase in twinning crystal fraction, the tensile strength of pre-charged hydrogen specimens increases from 557 MPa to 576 MPa and 694 MPa sequentially, indicating that pre-straining can significantly enhance the tensile strength of high-manganese steel. Meanwhile, the subsurface hydrogen concentration decreases from 8.323 mg/L to 5.206 mg/L and 3.362 mg/L with the increase in twinning crystal fraction. The improvement of twinning crystal fraction effectively enhances the hydrogen embrittlement resistance, reducing the hydrogen embrittlement susceptibility from 35.1% to 3.72% and 1.25% in turn. As the twinning crystal fraction increases, the twinning crystal lamella spacing shortens from 1.447 2 μm to 0.934 9 μm, which reduces the free path of dislocation motion during deformation, inhibits the diffusion and aggregation of hydrogen inside the microstructure, and alleviates the phenomenon of microvoid aggregation and local strain, thereby decreasing the probability of crack nucleation. Ultimately, this synergistically achieves the simultaneous improvement of strength and hydrogen embrittlement resistance of high-manganese steel.