Background: The role of PPARα in gene regulation in mouse liver is well characterized. However, less is known about the role of PPARα in human liver. The aim of the present study was to better characterize the impact of PPARα activation on gene regulation in human liver. To that end, chimeric mice containing hepatocyte humanized livers were given an oral dose of 300 mg/kg fenofibrate daily for 4 days. Livers were collected and analyzed by hematoxilin and eosin staining, qPCR, and transcriptomics. Transcriptomics data were compared with existing datasets on PPARα activation in normal mouse liver, human primary hepatocytes, and human precision cut liver slices. Results: Of the different human liver models, the gene expression profile of hepatocyte humanized livers most closely resembled actual human liver. In the hepatocyte humanized mouse livers, the human hepatocytes exhibited excessive lipid accumulation. Fenofibrate increased the size of the mouse but not human hepatocytes, and tended to reduce steatosis in the human hepatocytes. Quantitative PCR indicated that induction of PPARα targets by fenofibrate was less pronounced in the human hepatocytes than in the residual mouse hepatocytes. Transcriptomics analysis indicated that, after filtering, a total of 282 genes was significantly different between fenofibrate- and control-treated mice (P < 0.01). 123 genes were significantly lower and 159 genes significantly higher in the fenofibrate-treated mice, including many established PPARα targets such as FABP1, HADHB, HADHA, VNN1, PLIN2, ACADVL and HMGCS2. According to gene set enrichment analysis, fenofibrate upregulated interferon/cytokine signaling-related pathways in hepatocyte humanized liver, but downregulated these pathways in normal mouse liver. Also, fenofibrate downregulated pathways related to DNA synthesis in hepatocyte humanized liver but not in normal mouse liver. Conclusion: The results support the major role of PPARα in regulating hepatic lipid metabolism, and underscore the more modest effect of PPARα activation on gene regulation in human liver compared to mouse liver. The data suggest that PPARα may have a suppressive effect on DNA synthesis in human liver, and a stimulatory effect on interferon/cytokine signalling.