Three-dimensional (3D) ultrasonography has been shown to be an accurate and appropriate tool for measurement of gallbladder volume in humans. Therefore, we applied this novel technique for the first time to study fasting and postprandial gallbladder volume in 10 healthy dogs and compared the results with those of 2-dimensional (2D) ultrasonography. Fasting gallbladder volumes determined by 3D ultrasonography were significantly higher than corresponding volumes determined by 2D ultrasonography (P<0.01). Additionally, gallbladder volumes were significantly decreased in the postprandial state compared with the fasting state using 3D ultrasonography (P<0.001), but 2D ultrasonography showed no significant difference (P=0.189). The Gallbladder contraction index was higher in 3D ultrasonography than 2D ultrasonography; however, it did not reach statistical significance (P=0.25). In conclusion, 3D ultrasonography was able to measure gallbladder volume in healthy dogs in this study. It is suggested that 3D ultrasonography can be used to accurately estimate gallbladder volume and contractility.