INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is characterised by accumulation of triglycerides and cholesterol within the liver and dysregulation of specific hepatic cytochrome P450 (CYPs) activity. CYPs are involved in the metabolism of endogenous and exogenous chemicals. Hepatic CYP activity is dysregulated in human studies and animal models of a Western diet (WD) or low birth weight (LBW) independently, but the additive effects of LBW and postnatal WD consumption are unknown. As such, the aim of this study was to determine the independent and combined effect of birthweight and postnatal diet on hepatic CYP activity in a guinea pig model. METHODS: LBW was generated via uterine artery ablation at mid gestation (term = 70 days gestation). Normal birthweight (NBW) and LBW pups were allocated either a control diet (CD) or WD at weaning. After 4 months of dietary intervention, guinea pigs were humanely killed, and liver tissue collected for biochemical and functional hepatic CYP activity analyses. RESULTS: Independent of birthweight, functional activity of CYP3A was significantly reduced in female and male WD compared to CD animals (female, P < 0.0001; male, P = 0.004). Likewise, CYP1A2 activity was significantly reduced in male WD compared to CD animals (P = 0.020) but this same reduction was not observed in females. CONCLUSION: Diet, but not birthweight, significantly altered hepatic CYP activity in both sexes, and the effect of diet appeared to be greater in males. These findings may have clinical implications for the management of NAFLD and associated co-morbidities between the sexes.