Background: The aim of this study was to investigate the relationships between levels of n-3 essential polyunsaturated fatty acids (n-3 PUFAs) and stable nitric oxide (NO) metabolites in the plasma of athletes. Methods: Highly trained cross-country skiers (males, n = 39) were examined. The fatty acid profile of the total plasma lipids was determined by gas chromatography. The plasma NO level was studied by a colorimetric method via reaction with Griess reagent. Results: A widespread deficiency of essential n-3 PUFAs in the plasma of athletes (more than 80% of the subjects) was demonstrated in association with an imbalance in the levels of nitrates (NO3) and nitrites (NO2). A lower value of n-3 linolenic acid in the plasma (0.21 mol/%) was associated with a NO3 level below the normal range (n-3 C18:3 and NO3 Rs = 0.461; p = 0.003). Higher levels of n-3 eicosapentaenoic acid (0.8 mol/%) were associated with a concentration of NO2 above the normal value (n-3 C20:5 and NO2 Rs = 0.449; p = 0.004). Conclusion: For the first time, the participation of essential n-3 PUFAs in the nitrite–nitrate pathway of NO synthesis in highly trained skiers was demonstrated.