The fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by direct binding of ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4 and palmitate-bound pFABP5 from the gentoo penguin (Pygoscelis papua) at 2.1, 2.2, and 2.3 Å resolutions, respectively. The pFABP4 and pFABP5 proteins comprise a canonical β-barrel structure with two short α-helices forming a cap region and fatty acid ligand binds in the hydrophobic cavity inside the β-barrel structure. The two linoleate-bound pFABP4 and palmitate-bound pFABP5 structures shows a different ligand-binding mode and a unique ligand-binding pocket caused by several sequence differences (A76/L78, T30/M32, underlining used to indicate pFABP4 residues). Structural comparison also shows a significantly different conformation change in the β3-β4 loop region (residues 57-62) of pFABP5 as well as flipped Phe60 residue (the corresponding residue in pFABP4 is Phe58). Moreover, a ligand-binding study using fluorophore displacement assays indicated that pFABP4 has a relatively strong affinity to linoleate compared with pFABP5. In contrast, pFABP5 clearly exhibits higher affinity for the palmitate compared with pFABP4. Conclusively, our high-resolution structures and ligand-binding study provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. To investigate mechanism of fatty acid transfer, we have carried out structural studies. As the first step toward its structural elucidation, we report the results of preliminary X-ray crystallographic experiments with pFABP4, pFABP4-Linoleate and pFABP5-Palmitate.