Recent in vitro transcriptomic analyses for short-chain per- and polyfluoroalkyl substances (PFAS) HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate) added to the weight of evidence supporting the peroxisome proliferator-activated receptor alpha (PPARα) activator-induced hepatocarcinogenesis mode of action (MOA) for HFPO-DA-mediated liver effects in rodents. Importantly, PPARα-mediated key events (KEs) including hepatocellular hypertrophy and proliferation that have been shown to occur prior to tumor development in this MOA are rodent-specific and likely not human-relevant. To further inform the MOA of HFPO-DA and evaluate other hypothesized MOAs, phenotypic and transcriptomic responses in wild-type (WT) and Pparα-null mice were investigated following short-term exposure to HFPO-DA or prototypical agonists of PPARα (GW7647), PPARγ (rosiglitazone), or cytotoxicity (acetaminophen). Phenotypic and transcriptomic assessment of mouse livers demonstrated a general lack of response to HFPO-DA or GW7647 exposure in Pparα-null but not WT mice. Conversely, rosiglitazone or acetaminophen elicited similar phenotypic and transcriptomic responses between genotypes demonstrating a lack of PPARα-dependence. In WT mice, HFPO-DA-mediated responses were similar to GW7647 but different from rosiglitazone or acetaminophen. Dose-dependent increases in liver weight, karyomegaly, and mitosis, as well as increased transcriptomic signaling related to PPARα activation and cell proliferation were observed in HFPO-DA and GW7647-exposed WT mice. The consistent phenotypic and transcriptomic signaling patterns between HFPO-DA and GW7647 in WT mice, and the lack of changes in Pparα-null mice, provide further support that HFPO-DA-mediated early KEs in mouse liver are PPARα-dependent, occur via the rodent-specific PPARα MOA, and therefore are not appropriate for use in human health risk assessment.