Connective tissue, one of the main components of peri-implant soft tissue, is key to the formation of the peri-implant mucosal seal and helping to prevent epithelial ingrowth. Rough surfaces (Rs), machined surfaces (Ms) or microgrooved surface (MG) are used in the neck area of commercially available titanium implants. In this paper, we aimed to evaluate the influence of surface topography of titanium substratum on connective tissue fibroblasts to gain a better understanding of this effect. Fibroblasts were cultured on titanium plates with Rs, Ms and MG. Adhesion cell number at day 3 was compared and protein distribution of both F-actin and vinculin was determined to observe cellular structure and adhesion. Cell adhesion strength was compared on each surface. At day 3, the number of fibroblasts attached on each substratum was in the order of MG ≈ Ms > Rs. Fibroblasts strongly expressed vinculin in the peripheral area on Ms and MG, and showed strong F-actin architecture. Decreased expression of vinculin and weaker continuity of F-actin were observed on Rs. Fibroblasts on MG were aligned along the grooves, with a significantly higher cell density, whereas cells on Ms and Rs had no clear orientation. The cell adhesion strength was significantly lower on Rs, and no significant difference was seen between MG and Ms. Both MG and Ms showed greater adhesion cell numbers and adhesion strength of fibroblasts when compared with Rs at day 3. The cell density on MG was greater than those on other substrata.