The purpose of this study was to determine the differentially expressed genes in human gingival fibroblasts (HGFs) cultured on titanium (Ti) substrata with topographies presenting microgrooves and acid-etched roughness. Microgrooves were fabricated with a truncated V-shape in cross-section at 15/3.5, 30/10, and 60/10 μm (width/ depth) by photolithography. Subsequent acid etching was applied to the entire surface of the fabricated Ti substratum to generate etched microgrooves and ridges (designated as E15/3.5, E30/10, and E60/10). Both smooth and acidetched- only Ti were used as controls (designated as NE0 and E0). Large-scale gene expression analyses were performed using differential display PCR, and the results were confirmed using RT-PCR and quantitative real-time PCR. Of the 21 genes with altered expression determined by differential display PCR and sequencing, we verified through RT-PCR that MTDH and TIMP1 were up-regulated and TGF-β1, TPM1, and VIM were down-regulated in the HGFs cultured on E60/10 versus NE0. We also confirmed, by quantitative real-time PCR, that MTDH and TIMP1 expression in HGFs on E60/10 was significantly up-regulated compared to HGFs on the other Ti substrata. This study indicates that acid-etched ridges and microgrooves on Ti with a width and depth of 60 and 10 μm (E60/ 10) induce alterations in the expression of genes involved in cell adhesion, proliferation, and regulation of the cytoskeleton in HGFs.