Maritime pine (Pinus pinaster)-seed oil contains two Delta5 unsaturated polymethylene interrupted fatty acids (all cis-5,9, 12-18:3 and all cis-5,11,14-20:3 acids) one of which resembles eicosapentaenoic acid. The goal of the present study was to test whether maritime pine-seed oil consumption affects HDL and apolipoprotein (Apo) A-I levels as well as the ability of serum to promote efflux of cholesterol from cultured cells. To this end, wild type (WT) non-transgenic mice and transgenic mice expressing human ApoA-I (HuA-ITg) were fed on isoenergetic diet containing either 200 g maritime pine-seed oil/kg or 200 g lard/kg for 2 weeks. WT and HuA-ITg mice fed maritime pine-seed oil had lower cholesterol, HDL-cholesterol, LDL-cholesterol and HuA-ITg mice had lower human ApoA-I than those fed lard. The differences in cholesterol (P < 0.0001) and HDL-cholesterol (P < 0.003) levels between mice fed on the two diets were more pronounced in the HuA-ITg than in the WT mice. The ability of HuA-ITg serum to promote cholesterol efflux in cultured cells was greater (P < 0.008) than that of WT animals. However, the maritime pine-seed oil diet was associated with lower (P < 0.005) in vitro cholesterol efflux ability than the lard diet in both mice genotypes. This suggests a negative effect of the maritime pine-seed oil on reverse cholesterol transport. Cholesterol efflux was correlated with serum free or esterified cholesterol and phospholipid levels. The slope of the regression line was smaller in the HuA-ITg than in the WT mice indicating that overexpression of human ApoA-I reduces the negative impact of maritime pine-seed oil on cholesterol efflux. In conclusion, maritime pine-seed oil diet lowers HDL-cholesterol and diminishes in vitro cholesterol efflux. This potentially detrimental effect is attenuated by overexpression of human ApoA-I in mice.