In this study we assessed whether conditioned media from a human pancreatic cancer cell line (MIA PaCa 2) can interfere with some intracellular pathways involved in glucose metabolism in isolated rat hepatocytes. The hepatocytes, isolated from Male Wistar rats, were incubated with MIA PaCa 2-conditioned or nonconditioned media. Conditioned and nonconditioned hepatocytes were run for 120 min in the presence or absence of insulin (100 mM) and were sampled at fixed time intervals. Supernatant glucose levels decreased to a similar extent over time in both conditioned and nonconditioned hepatocytes, while lactate levels significantly increased in nonconditioned hepatocytes with respect to conditioned hepatocytes. A pyruvate kinase activity increase was observed only in nonconditioned hepatocytes and was biphasic in nature, since this increased activity was detected both after a few and after 30 min following insulin stimulation. The cyclic AMP level increase was significantly higher in conditioned than in non-conditioned hepatocytes. It appears that MIA PaCa 2 cells produce a factor(s) that may interfere with one of the insulin-mediated intracellular pathways of glucose metabolism, namely, glycolysis. This detrimental effect on glycolysis is supported by the blunted rise in lactate concentration in the medium after the glucose challenge. This substance(s) probably transfers its signal inside the target cells, activating the adenylate cyclase pathway. These results support the hypothesis that pancreatic cancer is the cause rather than the consequence of diabetes mellitus.