The value of contrast-enhanced ultrasound (CEUS) in differential diagnosis between benign and malignant gallbladder diseases was investigated. Thirty-three patients with gallbladder carcinomas and 47 with benign gallbladder diseases underwent CEUS. The lesion enhancement time, enhancement extent, pattern, dynamic change of enhancement and the intactness of gallbladder wall were evaluated. In the early phase at CEUS, hyper-, iso-, hypo-, and non-enhancement were found in 84.8% (28/33), 9.1% (3/33), 6.1% (2/33), and 0% (0/33) of gallbladder carcinomas, and 70.3% (33/47), 17.0% (8/47), 2.1% (1/47), and 10.6% (5/47) of benign diseases (p > 0.05). Hyper-enhancement or iso-enhancement in the early phase and then fading out to hypo-enhancement within 35 s after contrast agent administration was found in 90.9% (30/33) of carcinomas and 17.0% (8/47) of benign lesions (p < 0.001). Destruction of the gallbladder wall intactness was absent in benign diseases, whereas it was present in 28 (84.8%) of the 33 carcinomas (p < 0.001). Destruction of gallbladder wall intactness on CEUS yielded the highest capability in differential diagnosis, with sensitivity, specificity, and Youden's index of 84.8% (28/33), 100% (47/47), and 0.85, respectively. Conventional US made correct original diagnoses in 55 (68.8%) patients, whereas CEUS in 77 (96.3%). Thus, CEUS is useful in differential diagnosis between malignant and benign gallbladder diseases.