Lipid droplet (LD) is surrounded by phospholipid monolayer mainly composed of phosphatidylcholine (PC). Steroidogenic acute regulatory protein (StAR)-related lipid transfer domain containing 10 (STARD10) has been shown to transfer PC between membranes in vitro. Lysophosphatidylcholine acyltransferase1 (LPCAT1) catalyzes the conversion of lysoPC to PC. The purpose of this study was to elucidate the role of STARD10 and LPCAT1 in LD formation. We hypothesized that the LD size depends on the surface-to-volume ratio. STARD10 and LPCAT1 were partly co-localized at the surface of LD in mouse hepatoma cells (Hepa1-6). The number of small LDs was increased by LPCAT1 overexpression, while the number of large LDs was increased by the overexpression of both STARD10 and LPCAT1. The percentage of small LDs was significantly higher in Stard10 knockout Hepa1-6 cells than that of normal Hepa1-6 cells. In the liver of Stard10 knockout mice, the total LD area was smaller and the sphericity of LD was lower than those of wild type mice, indicating that surface-to-volume ratio was higher. These results indicate that STARD10 and LPCAT1 are involved in LD formation by regulating surface-to-volume ratio through its activity of PC transfer and synthesis.