Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were firstly treated with a single intraperitoneal (IP) injection of rapamycin or vehicle and then submitted to a second IP administration of glucose 4 h after. Our results revealed that IP administration of glucose induced hyperglymia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish between 8 and 17 h and displayed a slightly increasing tendency whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt. Despite no significant variation was observed for phosphoenolpyruvate carboxykinase (mPEPCK) mRNA abundance, gene expression of glucokinase (GK), glucose 6-phosphatase (G6Pase) I, G6Pase II and fructose 1,6-bisphosphatase (FBPase) was lowered by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppressions of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1 dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout.