Dietary very long chain omega (omega)-3 polyunsaturated fatty acids (PUFA) have been associated with reduced CVD risk, the mechanisms of which have yet to be fully elucidated. LDL receptor null mice (LDLr-/-) were used to assess the effect of different ratios of dietary omega-6 PUFA to eicosapentaenoic acid plus docosahexaenoic acid (omega-6:EPA+DHA) on atherogenesis and inflammatory response. Mice were fed high saturated fat diets without EPA and DHA (HSF omega-6), or with omega-6:EPA+DHA at ratios of 20:1 (HSF R=20:1), 4:1 (HSF R=4:1), and 1:1 (HSF R=1:1) for 32 weeks. Mice fed the lowest omega-6:EPA+DHA ratio diet had lower circulating concentrations of non-HDL cholesterol (25%, P<0.05) and interleukin-6 (IL-6) (44%, P<0.05) compared to mice fed the HSF omega-6 diet. Aortic and elicited peritoneal macrophage (Mphi) total cholesterol were 24% (P=0.07) and 25% (P<0.05) lower, respectively, in HSF R=1:1 compared to HSF omega-6 fed mice. MCP-1 mRNA levels and secretion were 37% (P<0.05) and 38% (P<0.05) lower, respectively, in elicited peritoneal Mphi isolated from HSF R=1:1 compared to HSF omega-6 fed mice. mRNA and protein levels of ATP-binding cassette A1, and mRNA levels of TNFalpha were significantly lower in elicited peritoneal Mphi isolated from HSF R=1:1 fed mice, whereas there was no significant effect of diets with different omega-6:EPA+DHA ratios on CD36, Mphi scavenger receptor 1, scavenger receptor B1 and IL-6 mRNA or protein levels. These data suggest that lower omega-6:EPA+DHA ratio diets lowered some measures of inflammation and Mphi cholesterol accumulation, which was associated with less aortic lesion formation in LDLr-/- mice.