We present the effect of nanotopographically defined surfaces on adipocyte differentiation using various nanogroove patterns. Parallel nanogroove arrays with equal inter-groove distance (400, 550, 800 nm width) and varying distances (550 nm width with three different spacings of 550, 1100, and 2750 nm) were fabricated by UV-assisted capillary force lithography (CFL) on 18 mm diameter glass coverslips using biocompatible polyurethane (PU)-based material. After coating with fibronectin and subsequent culture of 3T3-L1 preadipocytes, the degree of adipocyte differentiation was determined by Oil Red O staining and adipogenic gene expression. We observed that adipocyte differentiation was slightly but substantially affected by culture on various nanogrooved surfaces. In particular, the cell crawling into nanogrooves contributed substantially to an enhanced level of differentiation with higher contact guidance, suggesting that cell-to-surface interactions would play a role for the adipocyte differentiation.