Apolipoprotein A-I (apoA-I) is a major component of high-density lipoprotein (HDL), which displays anti-atherosclerotic activity in plasma. In the current study, we compared the functional and structural characteristics of human, bovine and porcine apoA-I as regards their antioxidant ability and protein stability. In the lipid-free state, the immunoreactivity of bovine and porcine apoA-I differed from that of human apoA-I and bovine and porcine apoA-I exhibited greater resistance to denaturation induced by urea treatment. Bovine apoA-I showed the weakest binding ability of dimyristoyl phosphatidylcholine; however, bovine apoA-I formed slightly larger reconstituted HDL (rHDL) particles with palmitoyl oleoyl phosphatidylcholine, with a higher number of apoA-I-containing particles. Bovine and porcine apoA-I comprised of pentameric structures, whereas human apoA-I in the rHDL state consisted of trimeric structures. Although apoA-I from all three species showed a similar content of α-helicity in the lipid-free state (approximately 53%), bovine apoA-I showed a lower α-helicity content (approximately 66%) compared with human apoA-I (approximately 74%) in the rHDL state. Bovine apoA-I was more resistant to denaturation and glycation upon treatment with urea and fructose, respectively. Furthermore, bovine apoA-I showed a greater inhibition of cupric ion-mediated low-density lipoprotein (LDL) oxidation and uptake of acetylated LDL by macrophages compared with human or porcine apoA-I in the lipid-free and lipid-bound states. In conclusion, bovine apoA-I has unique functional properties in the lipid-free and lipid-bound states, and displays significantly enhanced anti-atherosclerotic activity.