Medical grade 316L stainless steel was laser surface melted (LSM) using continuous wave Nd-YAG laser in argon atmosphere at 1 and 5 mm/s. The treated surfaces were characterized using electron backscatter diffraction to study the influence of top surface crystallographic orientation and type of grain boundaries on corrosion resistance, wettability, and biocompatibility. The laser scan velocity was found to have a marginal influence on the surface roughness and the type of grain boundaries. However, the crystal orientation density was found to be relatively high in 1 mm/s samples. The LSM samples showed a higher concentration of {101} and {123} planes parallel to the sample surface as well as a higher fraction of low-angle grain boundaries. The LSM samples were found to exhibit better surface wettability and enhanced the viability and proliferation of human fetal osteoblast cells in vitro when compared to the untreated samples. Further, the corrosion protection efficiency of 316L stainless steel was improved up to 70% by LSM in as-processed condition. The increased concentration of {101} and {123} planes on surfaces of LSM samples increases their surface energy, which is believed to be responsible for the improved in vitro cell proliferation. Further, the increased lattice spacing of these planes and high concentration of low-energy grain boundaries in LSM samples would have contributed to the better in vitro corrosion resistance than untreated 316L stainless steel. Our results indicate that LSM can be a potential treatment option for 316L stainless steel-based biomedical devices to improve biocompatibility and corrosion resistance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 569–577, 2018. © 2017 Wiley Periodicals, Inc.