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To explore the feasibility of using diffusion tensor imaging (DTI) in the diagnosis of anal fistula and evaluating its activity. Thirty-four patients with perianal fistulas were examined with DTI on a 3.0 T magnetic resonance imaging (MRI) before undergoing surgery. Based on the surgery requirement and preoperative examinations, the lesions fell into 2 groups: the positive inflammation activity (PIA) group and the negative inflammation activity (NIA) group. Each lesion was divided into 3 regions of interest (ROIs) (i.e., the fistula area, edema area, and distant normal-appearing area). Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values were calculated and analyzed. There were statistically significant differences in FA and ADC values of the fistula area, edema area, and distant normal-appearing area. The FA values of the fistula area, edema area, and distant normal-appearing area in PIA were 0.134 ± 0.046, 0.225 ± 0.060, 0.343 ± 0.070, respectively. The ADC values (×10−3 mm2/s) of the fistula area, edema area, and distant normal-appearing area in PIA were 0.979 ± 0.441, 1.542 ± 0.274, 1.864 ± 0.336, respectively. The FA values of the fistula area, edema area, and distant normal-appearing area in NIA were 0.183 ± 0.057, 0.286 ± 0.059, 0.382 ± 0.084, respectively. The ADC values (×10−3 mm2/s) of the fistula area, edema area, and distant normal-appearing area in NIA were 1.393 ± 0.256, 1.518 ± 0.274, 1.703 ± 0.432, respectively. Regarding the activity, the FA and ADC values of the PIA group were lower than those of the NIA group in the fistula area, and the differences were statistically significant (P = .009, .004). The FA values of the edema area in the PIA group were lower than those in the NIA group, and the difference was statistically significant. The ADC values of the edema area, and both the FA and ADC values of the distant normal-appearing area all exhibited no statistically significant differences between the 2 groups. DTI parameters may reflect microstructures of perianal fiatulas via quantitative information. FA and ADC values were instrumental in evaluating the activity of perianal fistulas.