The effects of different pre-strain levels on the dislocation density, twinning behavior, resultant tensile properties, and cryogenic impact toughness of a high-manganese austenitic steel for low-temperature service were investigated. The results indicate that the dislocation density and volume fraction of twins are sharply increased when the pre-strain exceeds 15%, leading to an increase in yield strength and a decrease in impact toughness. At a 5% pre-strain level, few mechanical twins are observed while the dislocation density increases, resulting in enhanced yield strength whilst maintaining the toughness. The dislocation and grain refinement strengthening effects dominate the yield strength at various pre-strain levels. The initial mechanical twins and increased dislocations induced by pre-straining adversely affect the impact toughness. These findings validate the potential of controlling the mechanical twins and dislocations via pre-strain treatment as an effective approach to tailoring the mechanical properties of high-manganese austenitic steel.