It is suggested that obestatin can stimulate the secretion of pancreatic juice in rats and this effect is abolished by vagotomy. Thus, the aim of the present study was to further elucidate the mechanism by which obestatin controls the exocrine pancreas secretion. Anesthetized male Wistar rats (200 ± 15 g body weight) were administered intravenously (iv) and intraduodenaly (id) every 30 min obestatin in boluses of 30, 100 and 300 nmol ∙ kg−1 body weight and 15 min later pancreatic-biliary juice (PBJ) was collected to determine the PBJ volume, total protein and enzymes activity. Obestatin injections were also done following subdiaphragmatic vagotomy, capsaicin deafferentation and pharmacological blockage of the mucosal cholecystokinin 1 (CCK1) receptor with tarazepide. Dispersed acinar cells were isolated from rat pancreas by collagenase digestion, stimulated with CCK-8 (10−10 M) and incubated with obestatin (10−9–10−6 M) in vitro. It was noted that iv and id obestatin administrations did not affect the PBJ volume but increased protein and trypsin outputs regardless way of administration, and amylase and lipase outputs after id injection. Similarly to vagotomy, the capsaicin and tarazepide pre-treatments abolished the effects of obestatin. In contrast to the in vivo experiment, the treatment of dispersed pancreatic cells with the CCK-8+obestatin combination showed that obestatin decreased the CCK-8-stimulated amylase release from acinar cells in vitro, but obestatin alone did not exert effect on amylase release. So, it is thought that obestatin can stimulate the exocrine pancreas secretion via an indirect vagal mechanism, whilst its direct action on the acinar cells is also possible but with the opposite effects.