Hepatic fatty acid (FA) composition, especially a reduction in n-3 polyunsaturated FA (PUFA) may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is common in HIV-infection.. In a cross-sectional study we compared hepatic FA composition between 20 HIV-infected men with NAFLD (HIV/NAFLD), 21 HIV-negative men with NAFLD (NAFLD), and 7 healthy controls. Within HIV/NAFLD we compared simple steatosis (HIV/SS) to steatohepatitis (HIV/NASH). FA composition in liver and erythrocytes, oxidative stress, diet, and exercise were assessed. Major findings (P<0.05) were: 1) higher hepatic n-6/n-3 ratio in HIV/NAFLD [median (range)] [8.08 (1.08-21.52)] compared to controls [5.83 (3.58-6.93)] and NAFLD [5.97 (1.46-10.40)], with higher n-6 PUFA in HIV/NAFLD compared to NAFLD; 2) lower n-3 PUFA in erythrocytes (mol%), a marker for dietary intake, in HIV/NAFLD [5.26 (1.04-11.75)] compared to controls [8.92 (4.79-12.67)]; 3) the ratios of long-chain PUFA products to essential FA precursors of the n-6 and n-3 series were lower in HIV/NAFLD and NAFLD compared to controls. In contrast, the ratio of oleic/stearic acid was higher in HIV/NAFLD compared to the other groups. These ratios are indirect markers of enzymatic FA desaturation and elongation. Hepatic PUFA, especially biologically active long-chain PUFA, were also lower in HIV/NASH compared to HIV/SS. Oxidative stress was not different among the groups. We conclude that HIV/NAFLD is associated with altered hepatic FA composition. Changes may be due to impaired FA metabolism or suboptimal n-3 PUFA intake. The potential role of n-3 PUFA (e.g. fish oil) to treat or prevent HIV/NAFLD warrants further investigation.