Utilizing rainbow trout (Oncorhynchus mykiss) as a known model of "glucose intolerant" and poor dietary glucose user, glucose utilization was assessed in fish receiving chronically two molecules able to ameliorate glucose homeostasis: insulin and metformin. Our objectives were to assess at molecular level the ability of rainbow trout to deal with a glucose load and to improve glucose utilization in fish receiving chronically insulin plus metformin treatments. Fish received (mini-osmotic implanted pumps) saline, insulin, metformin and insulin plus metformin solution for 4 days and then were subjected to a glucose challenge (intraperitoneal injection) in order to study glucose homeostasis, analyzing plasma glycemia, glucose-related gene expression, insulin signalling and glycogen levels in liver and muscle. Control fish received saline pump implantation and saline intraperitoneal injection. We found no evidences that the glucose intolerance in this species could be based on molecular impaired metabolism in the tissues assessed. By contrast, very interestingly, we show for the first time, that metformin is not only unable to improve glucose homeostasis in trout, but also that counteract insulin effects, creating insulin resistance, especially in the muscle. These results make trout an attractive original model to study both insulin and metformin effect on biological systems.