Compared to fishmeal and fish oil, plant ingredients differ not only in the protein content, amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. In order to investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1g body weight during six months with a totally plant-based diet (V), a marine diet (M) and a marine restricted diet (MR) with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were up-regulated in trout fed V diet whereas expression of genes related to bile acid synthesis (cyp7a1) and cholesterol elimination (abcg8) were reduced. Feeding trout with V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα and miR-223. However, plasma and body cholesterol levels in trout fed V diet were lower compared to fish fed marine diet raising the question of the role of cholesterol in the negative effect of plant based-diet on growth.