In the context of fish oil replacement by alternative plant-derived oils in the fish feed, the influence of lignans, which are phenolic compounds present at significant concentrations in some plant-derived oils, was investigated on the omega-3 (n-3) fatty acid metabolism of the rainbow trout liver cell line RTL-W1 enriched in alpha-linolenic acid (ALA, 18:3n-3). The major linseed oil lignans, namely secoisolariciresinol diglucoside (SDG) and secoisolariciresinol (SECO), as well as the enterolignan enterodiol (END) were evaluated. Moreover, the effects of sesame oil lignans, namely sesamin (SES) and episesamin (EPI), were also investigated and compared to those previously observed in in vivo and in vitro studies which reported favourable effects on the fatty acid bioconversion capacity of salmonids. Cells were incubated with 50 μM ALA and 50 μM of SES, EPI, SDG, SECO or END for 48 h at 19 °C. The cells incubated with ALA had significantly higher amounts of 18:4n-3, 20:4n-3 and 20:5n-3 (eicosapentaenoic acid, EPA) as compared to control cells. The supplementation with SES decreased the 18:4n-3, 20:4n-3 and EPA contents, which indicates an inhibition of the desaturation and elongation steps involved in the fatty acid bioconversion pathway. As compared to SES supplementation, EPI supplementation showed a limited impact by only reducing 18:4n-3 and 20:4n-3. Similarly, END supplementation decreased 18:4n-3 and EPA, suggesting an inhibition of the desaturation capacity. Conversely, the supplementation of linseed lignans SDG and SECO had no impact on the fatty acid bioconversion capacity of RTL-W1 liver cells. In conclusion, the present results on the sesame seed lignans SES and EPI contradict the positive effects previously reported on fish lipid metabolism. In contrast, no influence of the linseed lignans SDG and SECO was observed. This suggests that the presence of lignans in linseed oil has no impact on the fish fatty acid bioconversion capacity when the oil is included to the diet.