In vitro 3D cultures of hepatocytes are increasingly being used to assess human hepatic metabolism and toxicity in drug development. Here, we developed an in vitro 3D cell culture method with a microstructured mesh sheet and applied it to culturing human hepatoma HepG2 cells. The micromesh sheet is constituted of fine mesh strands and apertures that are each much larger than a single cell in size. Proliferating on a micromesh sheet, HepG2 cells spread out in a planar manner and then formed a multilayered cell sheet, so that cell-cell adhesion was dominant over cell-substrate adhesion as being different from 2D cultures. In micromesh cultures, the increase rate in thickness of the cell mass was visually slower than that in spheroid cultures, enabling us to clearly observe inside cells of the cell population by microscopy. Micromesh-cultured HepG2 cells showed higher viability compared with spheroid-cultured cells. The multilayered HepG2 cell sheet increased expression of hepatic marker genes and induced cell polarization with bile canalicular membranes. Furthermore, a combination of micromesh cultures with medium perfusion further induced expression of hepatic marker genes in HepG2 cells; especially CYP1A1 and CYP1A2 mRNA increased 86-fold and 43-fold compared with 2D controls, respectively, which were much higher than those in spheroid cultures. Thus, this simple and versatile micromesh culture method holds some advantages over traditional spheroid cultures and is expected to be instrumental in culturing more differentiated hepatic cells such as HepaRG cells and primary hepatocytes for future preclinical testing.