In this study, we assessed the binding affinities of perfluoroalkyl substances (PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs), to the ligand-binding domains (LBDs) of Baikal seal ( Pusa sibirica; bs) and human (h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs) of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA > PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation with those to hPPARα. In silico PPARα homology modeling predicted that there were two ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity relationship analyses suggested that the binding potencies of PFASs to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains than hPPARα. The in silico docking simulations suggested that the first LBP of bsPPARα had higher affinities than that of hPPARα; however, the second LBP of bsPPARα had lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing interspecies differences in the binding of PFASs to PPARαs and their structure-activity relationships.