Non-alcoholic fatty liver disease (NAFLD), characterized by triglyceride deposition in hepatocytes due to imbalanced lipid homeostasis, is of increasing concern in Western countries, with progression to non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 μM each), and/or with 10 ng/mL of the inflammatory mediator interleukin-6. Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane-X-receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. Interleukin-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors, rather than to reduced transcription of nuclear receptor-encoding mRNAs. Most striking effects of interleukin-6 and/or fatty acid treatment were observed in HepaRG cells following 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation and xenobiotic-induced drug metabolism.