Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. We used a NanoCulture plate to determine the spheroid-forming ability of pancreatic ductal adenocarcinoma (PDAC) cell lines and compared the morphology and expression of cytoskeletal proteins of PDAC cells to those in two-dimensional (2D) cultures. All examined PDAC cells grew as monolayers in 2D culture. PANC-1 and KLM-1 formed spheroids in 3D culture, but PK-45H and MIAPaCa-2 did not. Strong expression of F-actin was observed in the cells attached to the surface of the plate, which formed cell projections in 3D culture. F-actin was detected on the grids of the NanoCulture plate in PANC-1 cells but not in PK-45H. The levels of tubulin expression in cells were higher in 3D culture than in 2D culture. The expression level of E-cadherin mRNA in PANC-1 and KLM-1 was higher than that in PK-45H and MIAPaCa-2. In conclusion, PDAC cells showed morphological changes, spheroid formation, and alterations of cytoskeletal proteins in 3D culture. E-cadherin might be one of the key molecules involved in spheroid formation of PDAC cells. The 3D spheroidal culture system was a useful method for cell imaging with contrast-phase microscopy and confocal microscopy.