The purpose of this study was to determine the influential factors on the early osteoblastic differentiation of MG63 osteoblast-like cells cultured on micro-grooved Ti substrata. Commercially pure Ti discs with surface microgrooves of various dimensions were fabricated using photolithography. Both ground and acid-etched Ti were used as controls. Surface widths of the Ti substrata were calculated and used as the surface area parameters. These surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA). Early osteoblastic differentiation of MG63 human osteoblast-like cells were analyzed using an alkaline phosphatase (ALP) activity test. Pearson`s correlation analysis and multiple regression analysis were used to analyze the correlations between experimental results and to determine the influential factors on osteoblastic differentiation, respectively. The results of experiments showed significant correlations between surface properties and osteoblastic differentiation. Surface width was determined to be the greatest influential factor on the ALP activity after 1 day of osteogenic culture. The water contact angles measured in perpendicular directions to the microgrooves showed greatest influence on the ALP activity on day 7. This study indicates that the factors influencing the early osteoblastic differentiation on micro-grooved Ti substrata show respective time-dependence.