Objective To retrospectively evaluate the value of MR diffusion-weighted imaging (DWI) in differentiating between benign and malignant gallbladder lesions. Methods Forty-six patients (21 malignancy and 25 benignancy) were reviewed. ADC was calculated using two DW images with different motion-probing gradient strengths (b=0, 800s/mm2 ). The ADC values of the lesions were measured from ADC maps. Statistical analysis was performed using independent-sample t test. Diagnostic accuracy was evaluated using the receiver operator characteristic method. Results The mean ADC value of cancer was (1.23±0.19)×10-3mm2 /s, which was significantly lower than of the benign lesions (1.93±0.33)×10-3mm2 /s (t=-8.57, P<0.05). In receiver operating characteristic curve analysis, the areas under the curves for ADC were 0.95. The optimal cutoff values of ADC for differentiating benign and malignant lesions of the gallbladder was 1.42×10-3 mm2 /s with sensitivity of 90.5%, specificity of 92.0% and accuracy of 91.3%. And ADC had a high consistency compared with pathological results (Kappa values were 0.825). Conclusion DWI can improve diagnostic accuracy for differentiating malignant from benign gallbladder lesions.