No evidence of premature vascular disease is found in apolipoprotein A-I Milano (apoA-I M) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I M may impart a “gain of function” in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I M were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I Hu/Hu and h-B/A-II/A-I M Hu/Hu). On a chow diet, h-B/A-II/A-I M Hu/Hu mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I Hu/Hu mice (35.65 ± 8.00 mg/dl versus 58.09 ± 13.50 mg/dl, respectively; p < 0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I M Hu/Hu and h-B/A-II/A-I Hu/Hu lines. h-B/A-II/A-I M Hu/Hu females had higher total cholesterol levels compared to h-B/A-II/A-I Hu/Hu females (895.08 ± 183.07 mg/dl versus 544.43 ± 116.42 mg/dl; p < 0.05) and developed larger atherosclerotic lesions (148,260 ± 78,924 μm 2 versus 54,132 ± 43,204 μm 2, respectively; p < 0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I M Hu/Hu and h-B/A-II/A-I Hu/Hu males (19,779 ± 6098 μm 2 versus 15,706 ± 13,095 μm 2; p = 0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human apoA-I M gene does not have protective advantage over that of the apoA-I gene.