OBJECTIVE The purpose of this study was to investigate the effect of titanium (Ti) surface microgrooves and anodic oxidation on the surface characteristics of titanium and the osteogenic activity of human periodontal ligament cells (PLCs) cultured on theses surfaces. DESIGN Mechanically ground Ti was used as the control substratum (NE0). Truncated V-shaped microgrooves, 60μm-wide and 10μm-deep in cross-sections, were created on the Ti substrata by photolithography (NE60/10). Anodically oxidized Ti (NE0AO) and anodically oxidized microgrooved Ti (NE60/10AO) were also prepared. Scanning electron microscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were performed for surface characterization. Cell proliferation assay, osteoblast differentiation assay, and quantitative real-time PCR analysis were performed to compare the osteogenic activity of PLCs on NE0, NE60/10, NEAO, and NE60/10AO. RESULTS A decrease in the microgroove-width of NE60/10AO compared to NE60/10 due to Ti oxide layer generation by anodic oxidation was detected with XRD and XPS. Cell proliferation, osteoblast differentiation, and osteo-related gene expression were enhanced on the NE60/10AO substrata compared with NE0, NE60/10, and NE0AO. CONCLUSIONS The combination of Ti surface microgrooves and subsequent anodic oxidation treatment synergistically upregulated osteo-related gene expression, despite showing limited ability to increase cell proliferation and osteoblast differentiation levels in PLCs.