This study was aimed to evaluate the effect of region of interest (ROI) on measurement of apparent diffusion coefficient (ADC) obtained in diffusion-weighted (DW) MR sequences between spinal tuberculosis and malignancy, selecting the more reasonable and effective measurement of ADC value. We collected the conventional MR sequences and diffusion-weighted imaging data of thirty-two patients with spinal tuberculosis or malignancy confirmed clinically and pathologically from August 2009 to June 2010. Using the conventional MRI as a guide, the signal intensity (SI) and ADC value were calculated by selecting single ROI and the same area multi-ROI within whole lesion at the largest slice of lesion respectively. Using ADC value defined by receiver operating characteristic (ROC) curve analysis as threshold, the sensitivity, specificity, and accuracy of ADC value for diagnosing spinal tuberculosis were compared. The results showed that the mean of ADC value at signal ROI was lower than that at the multi-ROI. There was no significant difference between the ADC value of single ROI and multi-ROI. The ADC values from spinal tuberculosis and spinal malignancy obtained in single ROI or multi-ROI showed significant differences. The sensitivity, specificity, and accuracy of ADC value for differentiating tuberculosis from neoplastic infiltration at signal ROI is lower than that at the multi-ROI. In conclusion, the ADC acquired multi-ROI can describe preferablywater molecular diffusion and is more useful in differentiating spinal tuberculosis from malignant bone marrow lesions.