Hepatocyte nuclear factor 1 alpha (HNF1α) is an important transcription factor involved in the regulation of several drug-metabolizing cytochrome P450 enzymes. The gene of HNF1α-antisense 1 is located next to the HNF1α gene on the same chromosomal locus and encodes HNF1α-AS1, a long non-coding RNA (lncRNA). The hypothesis of this study is that the lncRNA HNF1α-AS1 is involved in the HNF1α-mediated regulation of the drug-metabolizing cytochrome P450s in liver cells. Knockdown of HNF1α protein or HNF1α-AS1 RNA was performed in HepaRG cells by small interfering RNA (siRNA) transfection. Expression of the selected P450s (including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) as well as their regulatory nuclear receptors of pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AHR), and hepatocyte nuclear factor 4 alpha (HNF4α) was quantified by RT-PCR and Western blot. Overnight transfection with HNF1α-siRNA produced a significant reduction in HNF1α mRNA and protein that lasted for 96 hours. Knocking down HNF1α in HepaRG cells dramatically depleted expression of the lncRNA HNF1α-AS1 RNA. Among the tested P450s, expression of CYP2C8, 2C9, 2C19, 2E1, and 3A4 was decreased, CYP1A2 was increased, and CYP2B6 and 2D6 did not change in the HNF1α knockdown cells, in which expression of PXR and CAR was also decreased, but AHR was increased and HNF4α didn’t change. Additionally, HNF1α knockdown attenuated P450 induction in HepaRG cells following treatment with phenobarbital (CAR activator) and rifampicin (PXR activator). Fold induction of CYP2C8, 2C9, 2C19, 2E1, 3A4 by phenobarbital and rifampicin was decreased in HNF1α knockdown cells. Knockdown of the lncRNA HNF1α-AS1 by siRNA did not affect expression of HNF1α and HNF4α, but showed similar effects on expression of the P450s as well as CAR and AHR as the HNF1α knockdown. These results suggest that HNF1α-AS1 lncRNA may play an important role in the HNF1α-mediated regulation of drug metabolizing P450s and nuclear receptors. A future study will determine how the lncRNA HNF1α-AS1 is involved in the HNF1α-mediated regulation of the P450 expression in liver cells.