The microstructure and fatigue and tensile properties of 316L stainless steel fabricated via laser powder bed fusion (L-PBF) were investigated. Two 316L stainless steel specimens with different loading directions which are either perpendicular to or parallel to building direction were prepared by L-PBF process. The results of X-ray diffraction tomography showed that there was no significant difference in morphology and size/distribution of the defects in the HB and VB samples. Since long axis of columnar grains is generally parallel to the build direction, the fatigue crack encounters more grain boundaries in VB samples under cyclic loading, which led to enhanced fatigue resistance of VB samples compared with HB sample. In contrast to HB sample, the VB sample has a higher fatigue strength due to a higher resistance to localized plastic deformation under cyclic loading. The differences in fatigue properties of L-PBF 316L SS with different build directions were predominantly controlled by solidification microstructures.