Abstract Purpose We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs). Materials and methods Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have 0/0 ㎛, 15/3.5 ㎛,30/10 ㎛, and 60/10 ㎛ of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at 700°C in air. The exper-iments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the one-way analysis of variance and Pearson’ s bivariate correlation analysis (SPSS Version 17.0). Results In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation. Conclusion Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.