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 biocompat-ibility 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 altative to biofunctionalized SLM-based Ti–6Al–4V implants with optimized physiochemical properties and biocompatibility for orthopedic and dental applications.