In this study, for the first time, the evolution of geometrically necessary dislocation (GND) and statistically stored dislocation (SSD) densities, as well as their roles in strain hardening during mechanical twinning, was experimentally investigated in a tensile-deformed Fe-22Mn-0.6C twinning-induced plasticity (TWIP) steel. GND and SSD densities were estimated via EBSD-acquired orientation data and a modified strain hardening model, respectively. The analysis demonstrates that the GND density increases non-linearly due to mechanical twinning. The SSD density increases much faster than the GND density, which shows that multiplication of the SSDs is heavily dependent on the imposed strain level. It is revealed that the GND density is higher at early strain stages (below 0.14 true strain), dominating dislocation hardening, but thereafter the SSD density contributes more. It is also found that the GND density is several times higher in this TWIP steel than in metals or alloys, which deform through dislocation slip only. We attribute this difference to the planar slip of dislocations and the occurrence of mechanical twinning, which leads to much more pile-ups of the GNDs at/near boundaries. Mechanical twinning directly contributes less than 100 MPa to flow stress increment in the studied true strain range of 0 to 0.34. Consequently, depending on dislocation types, dislocation multiplication governs strain hardening at all deformation ranges. The findings provide insight into the evolution behaviors of GNDs and SSDs in TWIP steels, which are particularly important for further understanding of the dynamic Hall-Petch effect and useful for TWIP alloy design efforts.