Background and Purpose: Recent in vitro transcriptomic analyses for the shortchain polyfluoroalkyl substance (PFAS), HFPO-DA (ammonium, 2,3,3,3-tetrafluoro2-(heptafluoropropoxy)-propanoate) (Klaren et al. 2023; manuscript in preparation), support conclusions from in vivo data that HFPO-DA-mediated liver effects in mice are part of the early Key Events of the peroxisome proliferator-activated receptor alpha (PPARα) activator-induced rodent hepatocarcinogenesis mode of action (MOA). Transcriptomic responses in HFPO-DA-treated rodent hepatocytes have high concordance with those treated with a PPARα agonist and lack concordance with those treated with PPARγ agonists or cytotoxic agents. The established PPARα MOA consists of four key events: 1) PPARα activation, 2) alteration in cell growth pathways, 3) perturbation of cell growth and survival, and 4) selective clonal expansion of preneoplastic foci cells. To further investigate Key Event 1 (PPARα activation) in the MOA for HFPO-DA in mouse liver and elucidate whether HFPODA-mediated transcriptomic responses in mouse liver are PPARα-dependent, an in vitro transcriptomic study was conducted using primary PPARα knockout (KO) and wild-type (WT) B6129SF2/J mouse hepatocytes. Methods: Whole-transcriptome templated oligomer sequencing (TempO-Seq) was conducted on samples from primary PPARα KO and WT mouse hepatocytes exposed for 12, 24 or 72 hours with various concentrations of HFPO-DA, or established agonists of PPARα (GW7647) and PPARγ (rosiglitazone), or hepatotoxicants (acetaminophen or d-galactosamine). Differentially expressed genes (DEGs), gene set enrichment and upstream regulator predictions, as well as dose-responsive genes and functional classification (i.e., pathway enrichment) of dose-responsive genes were determined for each hepatocyte genotype by chemical treatment group and timepoint. Results: Whole transcriptomic analyses of primary WT and PPARα KO mouse hepatocytes demonstrated nearly identical transcriptomic signaling pathways and predicted upstream regulators in both hepatocyte genotypes treated with HFPO-DA or the established PPARα agonist, GW7647. Pathways related to fatty acid metabolism and PPAR signaling were among the most significantly enriched for both chemicals. However, responses in PPARα KO hepatocytes were weaker, and exhibited a distinct temporal and concentration-dependent delay for both chemicals that did not occur in WT hepatocytes. Evidence for this delay was based on a low number of DEGs and general lack of enriched gene sets at 12 h, as well as an approximately 10-fold difference between benchmark concentrations (BMCs) for PPARα target genes and associated pathways in WT and PPARα KO hepatocytes at 24 and 72 h. Conversely, this delay was not observed in PPARα KO hepatocytes treated with the established PPARγ agonist, rosiglitazone, or known cytotoxic agents, acetaminophen or d-galactosamine. Conclusions: The similarity in transcriptomic signaling between HFPO-DA and GW7647 in both the presence and absence of PPARα in vitro supports the PPARα-dependence of HFPO-DA effects and further informs the transcriptomic responses of PPAR activators in the absence of PPARα. PPAR signaling and fatty acid metabolism-related pathways are mediated more efficiently in WT hepatocytes treated with HFPO-DA and GW7647, however, similar transcriptomic signaling, likely mediated by compensatory responses, was also observed in the absence of PPARα, albeit weaker and delayed. Follow-up in vivo studies in PPARα KO mice are needed to confirm PPARα dependence of HFPO-DA and to address the downstream rodent-specific key events of the PPARα MOA.