Functional bile canaliculus formation in cultured human hepatocytes is crucial for in vitro studies of hepatobiliary disposition and drug-induced cholestasis. Human hepatocytes isolated from humanized mouse livers (PXB-cells) are promising cell sources for these studies. PXB-cells available in tissue culture flasks allow users to recover and reseed in different cell culture formats, thereby enhancing their adaptability to various in vitro culture systems. However, the reseeding process may induce cellular stress, affecting subsequent cultures, and its specific effect on bile canaliculus formation is yet to be explored. Furthermore, the role of sufficient oxygen supply in bile canaliculus formation in PXB-cells remains incompletely understood. In this study, we compared hepatic function and bile canaliculi formation in freshly seeded PXB-cells (Fresh PXB-cells) and reseeded PXB-cells (Flask-delivered PXB-cells) under sufficient oxygen supply through oxygen-permeable plates. The flask-delivered PXB-cells recovered their levels of albumin production and cytochrome gene expression to those of fresh PXB-cells after seven days of culture. On days seven and 14 of culture, bile canaliculus formation was similar in both fresh and flask-delivered PXB-cells, as confirmed by fluorescein-labeled bile acid excretion and immunostaining for the bile canaliculi marker MRP2. Notably, analysis of bile canalicular length revealed a significant increase in bile canalicular length with adequate oxygenation, whereas no significant difference was detected between the conditions under the same oxygen supply on days seven and 14. The findings of this study provide valuable insights into the use of PXB-cells for in vitro assessments in drug discovery and toxicological research.