We reported the enhanced liver-specific function and structure of HepG2 cells by oxygenation culture via a collagen vitrigel membrane (Oshikata-Miyazaki and Takezawa, 2016). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. The aim of the present study is to clarify the features of HepG2-NIAS cells by comparing to HepG2 cells with two different cell culture histories. HepG2-NIAS cells subjected to oxygenation culture grew as a monolayer and showed a high CYP3A4 activity which was equivalent to almost half level to that of differentiated HepaRG cells and potential to form bile canaliculus-like structure. On the other hand, HepG2-RCB1648 cells and HepG2-RCB1886 cells subjected to oxygenation culture formed large multicellular aggregates and almost no detectable CYP3A4 activity and potential to form bile canaliculus-like structure. Protein expression levels of sinusoidal drug uptake and canalicular transporters were notably higher in HepG2-NIAS cells than HepG2-RCB1648 cells and HepG2-RCB1886 cells subjected to oxygenation culture. In contrast, protein expression levels of sinusoidal drug efflux transporters were highest in HepG2-RCB1648 cells. In conclusion, it is expected that HepG2-NIAS cells subjected to oxygenation culture are useful for predicting drug metabolism and excretion in the liver.