Plastic deformation of 3D printed components may occur when they are in use. Here we analyze the effects of the initial 3D printed microstructure of 316 L stainless steel on the subsequent deformation behavior, where we apply 10% and 30% thickness reductions by cold rolling. The microstructures are characterized by electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). Compared to conventionally manufactured (solution treated) samples, deformation twinning is observed to occur at lower strains in 3D printed samples, and twins are observed to be thinner and intersecting inside some grains. These observations are linked to the pre-existing dislocation structure in the 3D printed samples, where dislocations of various Burgers vectors facilitate deformation twinning. Furthermore, cells with an average size of 125 nm are observed to form inside the initial cellular structure. The strength calculated based on microstructural parameters generally agrees with experimental results, showing a large strengthening contribution from twin–matrix lamellae. The present study provides fundamental ideas for microstructural engineering of 3D printed metals for even better mechanical properties.