The most essential tools for studying drug hepatotoxicity, liver diseases, and bioartificial livers have always been models that can recapitulate liver physiology in vitro. The liver progenitor cell line HepaRG represents an effective surrogate of the primary hepatocyte. However, the differentiation of HepaRG relies on long-term induction using a high concentration of dimethyl sulfoxide (DMSO), which may compromise the research of drug metabolism and restrict the applicability of this hepatic model. Here, we present a novel hepatic maturation medium (HMM) for the differentiation of HepaRG, which is based on a cocktail of soluble molecules that mimick the in vivo environment. We showed that HMM could rapidly (about nine days) induce HepaRG differentiation into polarized hepatocytes with maturely metabolic functions. In addition, under three-dimensional culture conditions, the hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes (PHH). Our work demonstrates the utility of HMM as an alternative to the DMSO-dependent differentiation protocol for HepaRG; moreover, these results facilitate the application of HepaRG.