The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of thediet, affects various cellular properties such as membrane structure and fluidity, energy metabolism andsusceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellularresponse to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicityinduced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has animpact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium.Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enrichingthe growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3),docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docos-apentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were thenchallenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i)the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA con-tent of the growth medium: major modifications were a high incorporation of the supplemented PUFA inthe cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellu-lar phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturatedfatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly protected theRTL-W1 cells against both methylmercury and cadmium; (iv) DHA enrichment significantly protectedthe cells against cadmium but not methylmercury; (v) AA and LA enrichment had no impact on the celltolerance to both methylmercury and cadmium; (vi) the abundance of 20:3n-6, a metabolite of the n-6 biotransformation pathway, in phospholipids was negatively correlated to the cell tolerance to bothmethylmercury and cadmium. Overall, our results highlighted the importance of the fatty acid supply onthe tolerance of fish liver cells to methylmercury and cadmium.