Glycerol is the by-product of biodiesel production and its utilisation in fish feed has recently gained relevance. As an important metabolic intermediate and structural component of triacylglycerol (TAG), it is still unclear whether its supplementation affects lipid utilisation and/or deposition in different tissues. Accordingly, a set of studies was aimed to evaluate how increasing levels of dietary glycerol (0, 2.5 and 5%) affect lipid synthesis in the liver, muscle and adipose tissue. After a growth trial with rainbow trout (8 weeks) and European seabass (6 weeks) fish were sampled at 6 and 24 h to assess mRNA levels of lipid metabolism-related enzymes. The remaining fish were subjected to a metabolic trial consisting of a 6-day residence in deuterated water ( 2 H 2 O) for metabolic flux calculations. This study stands as the second part of a broader experiment that also aimed at evaluating the carbohydrate metabolism ( Viegas et al., 2022 ). Dietary supplementation at 5% glycerol significantly increased plasma TAG levels in both species and liver TAG levels in seabass, with no repercussions on the perivisceral fat index. Despite responding as expected in a postprandial setting, only fas and Δ6-fad in trout and Δ6-fad in seabass responded significantly by increasing with the dietary supplementation of glycerol. In trout, the observed differences in the regulation of these enzymes were not reflected in the de novo lipogenic fluxes. The fractional synthetic rates (FSR) were overall low in all tissues with an average of 0.04, 0.02 and 0.01% d –1 , for liver, muscle and perivisceral fat, respectively. In seabass on the other hand, and despite increased mRNA levels in Δ6-fad, the overall lipid profile in the liver muscle and perivisceral fat was higher in MUFA at the expense of lower PUFA. Moreover, glycerol supplementation altered the lipogenic capacity of seabass with hepatic fractional synthetic rates for TAG-bound FA increasing with increasing glycerol levels (0.32 ± 0.18, 0.57 ± 0.18, and 0.82 ± 0.24 for 0%, 2.5% and 5% glycerol supplementation, respectively). The findings of the present study suggest that supplementation up to 2.5% of glycerol did not severely impact the lipid metabolism nor increased lipogenic potential in liver, muscle and perivisceral fat accumulation.