Nutritional support in critically ill patients is a fundamental principle of patient care. Little is known about gallbladder motility during the interdigestive phase and in response to enteral feeding. The objective of this study was to investigate the effect of enteral feeding on gallbladder function in dogs. The cholagogue meal (Lipofundin infusion) was applied in four anatomical positions (jejunum, duodenojejunal junction, descending duodenum, stomach) in five healthy Beagle dogs. Gallbladder volume (GBV) was monitored by ultrasonography. Lipofundin infusion given through the feeding tube caused a maximal gallbladder contraction of 9.2% (range 3.7–13.9%) in the jejunum, 16.5% (9.1–22.1%) at the duodenojejunal junction and 26.3% (22.8–29.5%) in the descending duodenum. When the cholagogue meal was given through the mouth, it caused a mean 33.8% (28.6–46.5%) maximum gallbladder contraction in the same animals. In conclusion, we can establish that the ultrasound-guided gallbladder emptying method proved to be a useful technique for monitoring the cholagogue effect of Lipofundin meal applied in different anatomical positions of the intestine. The deeper the position of application, the smaller and shorter gallbladder contraction was evoked.