<p>The human HepaRG^™ cell line is the closest surrogate to primary culture of hepatocytes (PHH) for toxicology studies. However, differentiated HepaRG^™ cells express low levels of the cytochrome P450 2D6 (CYP2D6) involved in the biotransformation of many drugs. Herein, progenitor HepaRG^™ cells were transduced using lentiviral particles encoding both human CYP2D6 and GFP proteins. The resulting transgenic HepaRG^™ cells stably expressed catalytically active CYP2D6 at levels close to those observed in PHH from rapid metabolizers and HepaSH™ hepatocytes. In CYP2D6 transgenic HepaRG^™ cells, tramadol was metabolized into both N- and O-desmethyl tramadol as seen in humans while parental HepaRG^™ cells produced only N-desmethyl tramadol. Following treatment with perhexiline, the CYP2D6 expressing HepaRG^™ cells exhibited higher IC₅₀ values and reduced mitochondrial damages compared to those found in parental cells. Transcriptomic analysis revealed that the expression of CYP2D6 did not significantly affect the cells’ ability to proliferate and differentiate or compromise key hepatocyte-specific functions. However, we identified a small number of genes, including <i>NXF3</i> and <i>TRIM63</i>, which were up-regulated in transgenic cells. Using CRISPR/Cas9-mediated knockdown of GFP and/or CYP2D6 sequences, we demonstrated that NXF3 mRNA and protein inductions were triggered by the lentiviral mRNA encoding GFP and CYP2D6 rather than by genomic transgene integration. Together, these findings establish CYP2D6-transgenic HepaRG™ cells as an optimized and reliable hepatocyte-like model for studying the metabolism and toxicity of CYP2D6 substrates. Our results also support the hypothesis that the NXF3 gene may be a marker of cellular response to the expression of a lentiviral chimeric mRNA.</p>