Surface microgrooves and acid etching on titanium (Ti) have been proposed to enhance various cell behaviors. In this study, surface hydrophilicity, protein adsorption, and alkaline phosphatase activity of osteoblasts were analyzed and compared between microgrooved Ti, Ti with microgrooves and further acid etching, smooth Ti, and acid-etched smooth Ti. Correlations between the results of each experiment were analyzed using Pearson's correlation analysis, and the influential factor on alkaline phosphatase activity was determined using multiple stepwise regression analysis. Among groups, the Ti substrata with microgrooves and subsequent acid etching showed significantly greater surface hydrophilicity and alkaline phosphatase activity compared with smooth Ti, whereas the Ti substrata with only microgrooves showed the greatest protein adsorption. Multiple stepwise regression analysis determined the surface hydrophilicity of Ti as the influential factor on alkaline phosphatase activity. This study indicates that surface microgrooves and acid etching on Ti substrata enhance surface hydrophilicity, leading to increased alkaline phosphatase activity.