The differential diagnosis of gallbladder (GB) cancer from inflammatory diseases in patients with a thickened GB wall is difficult, whereas the pre-operative diagnosis of GB cancer is critical for selecting the appropriate surgical modality. We evaluated the clinical usefulness of endoscopic ultrasonography (EUS) for the differential diagnosis of GB wall thickening. The medical records, post-operative pathology reports, and images of EUS for 134 patients with GB wall thickening (>3 mm) who underwent laparoscopic or open cholecystectomies at our institution between December 2006 and February 2010 were retrospectively reviewed. Thirteen patients (9.7%) had neoplastic GB wall thickening (11 with adenocarcinomas and two with adenosquamous carcinomas) and the remaining 121 patients (90.3%) had non-neoplastic GB wall thickening (117 with inflammatory GB wall thickening and four with adenomyomatosis). The mean (± standard deviation [SD], mm) GB wall thickness was 6.5 ± 3.3 and 19.4 ± 7.7 for non-neoplastic and neoplastic GB wall thickening, respectively (P < 0.01). The EUS variables which had a statistically significant association with neoplastic GB wall thickening were GB wall thickening >10 mm, disruption of the normal two layers of the GB wall, hypoechoic internal echogenecity, and the absence of gallstones. Based on the multivariate analyses, GB wall thickening >10 mm and hypoechoic internal echogenecity were independent predictive factors for neoplastic GB wall thickening. The diagnostic power of EUS for differentiating neoplastic and non-neoplastic GB wall thickening could be improved by the appropriate application of some EUS variables.