TWIP steel as the representative of advanced high strength steel (AHSS) has a bright future in market and application owing to its excellent strength and ductility. Hydrogen embrittlement (HE) as a difficult problem of TWIP steel, researches on solving it mainly focus on alloying, few effort has been made on the mechanism of improving HE resistance by process adjustment and microstructure optimization. In this work, electrochemically combined with slow strain rate tensile test (SSRT) and OM, SEM have been used to study the effect of annealing temperature on mechanical property and deformation behavior of a Fe-18Mn-0.6C (mass fraction, %) twinning-induced plasticity (TWIP) steel, and also the influence of various microstructures on HE were discussed. The results showed that the grain size of TWIP steel increased with the increasing annealing temperature. In 700 ℃ annealed sheet, grain boundary (Fe, Mn)₃C cementite was obvious. TWIP steel with uniform medium-sized grains by 900 ℃ annealing had the highest strength-ductility balance. After SSRT under ongoing hydrogen charging, strength and plasticity reduced significantly. The strength-ductility balance loss rate (R) showed a tendency of increasing with the increasing annealing temperature. Deformation twins were more likely to be produced in large-sized grains by high temperature annealing. The junctions of twin/twin and twin/grain boundary were the main sources of hydrogen induced cracks. Although relatively low temperature annealing resulted in fine grains and the change of stacking fault energy (SFE) along grain boundary, deformation twins were not easily formed. But it was very vulnerable to generate vacancies where the stress concentrated between the local coarse carbides and deformation twins, then evolved into crack sources. As a result, the plasticity of TWIP steel itself was not high when annealed at a relatively low temperature. It had no apparent effect on improving HE resistance for TWIP steel annealed below 800 ℃.