Background <p>Studies evaluating the longitudinal relationship between plasma free fatty acids (FFAs) and subclinical atherosclerosis remain limited. We investigated the prospective association between serum FFA levels and the progression of coronary artery calcification (CAC) in a generally healthy, age-, sex-, and racially/ethnically diverse population.</p> Methods <p>This study utilized baseline data from the Multi-Ethnic Study of Atherosclerosis cohort, collected from 2000 to 2002, including 2988 women and 2696 men, with outcome data extending to 2012. CAC progression was defined as either the new onset of detectable CAC among participants with a baseline score of zero or a clinically meaningful increase in CAC severity among those with pre-existing CAC, based on established cut-offs. We employed Cox proportional hazards regression to calculate hazard ratios (HR) and 95% confidence intervals (95%CI) for the associations between FFAs and the CAC progression, stratified by sex. We evaluated potential non-linear relationships using restricted cubic splines.</p> Results <p>During the 9-year follow-up, we identified 1302 cases of CAC progression in women and 1480 cases in men. Serum FFAs were linearly associated with CAC progression in men, with a multivariable-adjusted HR of 1.41 (95% CI: 1.03–1.93) for each mmol/L increase in FFAs. Moreover, men in the highest relative to the lowest quintile of FFAs had a 20% higher risk (1.20; 1.02–1.43). Among women, we found evidence of a potential nonlinear association between FFAs and CAC progression, suggesting a threshold effect in their relationship (P non-linearity: 0.050). We observed no significant effect modification by age, body mass index, diabetes status, or hypertension in the relationship between FFAs and CAC progression.</p> Conclusions <p>This study highlights a significant association between FFAs and CAC progression in both men and women, though the nature of this association differs by gender.</p>