Deformation twinning in high-nitrogen austenitic Fe–18Cr–18Mn–2Mo–0.9N stainless steel is investigated in terms of orientation dependence and formation mechanism. The deformed microstructure is characterized by a planar dislocation structure in the low strain region and by stacking faults together with well-developed deformation twinning in the high-strain regime. The deformation twinning has a {111} 〈112¯〉 crystallographic component and shows strong orientation dependence with respect to tensile axis: (i) primary and conjugate twinning system cooperate in the 〈111〉 grain; (ii) only one twinning system is activated in the 〈110〉 grain; (iii) no deformation twinning is observed in the 〈100〉 grain. At the early stage of deformation, fault pairs composed of stacking fault planes and bounding partial dislocations heterogeneously nucleate and grow into overlapping stacking faults, resulting in the formation of deformation twinning. Based on the invisibility criteria using two-beam dynamical theory, the twinning partials are confirmed to be a Shockley dislocation with Burgers vector 16[12¯1], and no other dislocation components such as Frank or stair-rod type are found. The formation mechanism of deformation twinning in the present study could be accounted for by the three-layer twin model proposed by Mahajan and Chin, and is discussed in comparison with other models.