Introduction: Higher concentrations of circulating free fatty acids (FFAs) are common in MASLD, but little is known about the role of their composition. Methods: Serum levels of FFAs were measured after an overnight fast in a representative sample of the general US population. They were divided into saturated (palmitic, stearic and butirric acid), mono-unsaturated (palmitoleic and oleic acid), Ω-6 poli-unsaturated (Ω-6 PUFA: linoleic and arachidonic acid) and Ω-3 PUFA (alfa-linolenic, docosanoic (DHA) and eicosapentenaoic (EPA) acid)). Explorative end-points were: insulin resistance, liver steatosis and liver fibrosis estimated using HOMA-IR, a fatty liver index (FLI) score ≥60 and FIB-4 ≥1.3, respectively. Logistic regression analyses were performed to assess their associations with different FFAs after adjustment for confounders. Results: A total of 2440 people were included in the study. Concentrations of all considered FFAs increased progressively across HOMA-IR quartiles. A similar trend was identified when the population was segregated according to FLI. In logistic regression analyses adjusted for age, sex, body mass index and race-ethnicity, higher levels of all FFAs were significantly associated with insulin resistance (HOMA-IR≥2.5, OR ranging 1.69-3.41 per μmol/l increase) and with an elevated FLI (OR ranging 1.57-16.8 per μmol/l increase) while a higher DHA/EPA ratio was associated with lower FLI (OR: 0.81, 95%CI: 0.70-0.94, p=0.009). Finally, DHA (OR: 0.49, 95%CI: 0.27-0.90; p=0.025) and alfa-linolenic acid (OR: 0.72, 95%CI: 0.53-0.98; p=0.037) were associated with a lower prevalence of elevated FIB4 (>1.3). Conclusions: This is a cross-sectional, large population study suggesting that circulating FFA levels are associated with insulin resistance and steatosis; it also provides initial epidemiologic evidence in humans that Ω-3 PUFA in particular are associated with lower risk of liver steatosis and fibrosis, supporting data obtained in animal models. Moreover, their circulating levels might be manipulated using nutritional strategies in clinical studies.