Objectives: While inorganic mercury is being gradually withdrawn from industry, environmental exposure to mercury is recognized as one of the greatest present toxicological problems. The aim of this study was to evaluate the effect of polyunsaturated fatty acids (PUFAs) supplementation on selected cardiovascular risk factors and the urinary mercury (Hg-U) concentration in workers occupationally exposed to mercury vapor. Material and Methods: Overall, 38 workers of an electrolyzer hall (Hg-U: 46.6±35.7 μg/g creatinine) and a control group of 60 employees not exposed to Hg (Hg-U: 4.3±15.5 μg/g creatinine) were included in a clinical cross-over study. Clinical and laboratory tests were carried out 4 times: before and after a 3-month period of PUFAs supplementation (1000 mg daily), then after a 3-month break, and then after another 3-month period of PUFAs supplementation. Results: The baseline heart rate (HR) and serum triglyceride levels were higher in the Hg-exposed workers than in the controls, whereas systolic blood pressure (SBP) and cholesterol (C) levels exceeded normal values in both groups. There was a positive correlation between high-density lipoprotein 3 cholesterol (HDL₃-C) and Hg-U levels. The PUFAs use was associated with a decrease in both HR and SBP. After the first stage of supplementation, a decrease in the Hg-U concentration was observed. In a multivariate logistic regression model, decreases in Hg-U were associated only with exposure to mercury; OR_ΔHg = 0.562 (95% CI: 0.323–0.979), p < 0.042. After the second 3-month period of PUFAs supplementation, a significant association between HDL₃-C and a Hg-U decrease was shown: OR_HDL3 = 1.222 (95% CI: 1.01–1.46), p < 0.033. Conclusions: In the workers exposed to mercury vapor, PUFAs supplementation led to some beneficial effects on HR and SBP. The first stage of supplementation was associated with a decrease in Hg-U in which HDL₃ metabolism probably plays an important role. Int J Occup Med Environ Health. 2021;34(4):551–64