Cell behaviors in three-dimensional spheroids are known to be different from those in monolayer cultures; however, very few studies have compared the characteristics of cell spheroids formed through different biomaterial-induced processes. This study investigated the mechanism of melanocyte spheroid formation by using membranes composed of two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol (PVA). Our findings revealed that different spheroid-forming processes occurred on the two biomaterials. Human melanocytes were provided by the cell bank of the Department of Dermatology, National Taiwan University Hospital. The cell viability was determined through the MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide; Sigma) colorimetric assay. The cell living rate was determined using the trypan blue exclusion test. The amount of fibronectin adsorbed was quantified through Western blot analysis. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Duncan's test, and p values <0.05 or <0.01 was considered significant. In the study, the melanocytes attached to, migrated on, and aggregated on the chitosan surface and then formed spheroids. By contrast, on the PVA surface, the melanocytes directly aggregated to form three-dimensional spheres in suspension. The proliferative ability and survival rate of the melanocytes were considerably higher on the chitosan membranes than on the PVA membranes. We concluded that only cell-cell interactions dominated in melanocytes seeded on the PVA membrane, whereas cell-cell and cell-substrate interactions occurred on the chitosan membranes and further enhanced cellular functions. The chitosan-induced spheroids could probably overcome the diffusion and assimilation of trophic factors.