Selective laser melting (SLM) is a promising technique capable of rapidly fabricating customized implants having desired macro-and micro-structures by using computer-aided design models. However, the SLM-based products often have non-equilibrium microstructures and partial surface defects because of the steep thermal gradients and high solidification rates that occur during the laser melting. To meet clinical requirements, a heat treatment was used to tailor the physiochemical properties, homogenize the metallic microstructures, and eliminate surface defects, expecting to improve the cytocompatibility in vitro. Compared with the as-printed Ti-6Al-4V substrate, the heat-treated substrate had a more hydrophilic, rougher and more homogeneous surface, which should promote the early cell attachment, proliferation and osseointegration. More importantly, a crystalline rutile TiO2 layer formed during the heat treatment, which should greatly promote the biocompatibility and corrosion resistance of the implant. Compared to the untreated surfaces, the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) on heat-treated substrates were significantly enhanced, implying an excellent cytocompatibility after annealing. Therefore, these findings provide an alternative to biofunctionalized SLM-based Ti-6Al-4V implants with optimized physiochemical properties and biocompatibility for orthopedic and dental applications.