Objective To observe and analyze the relationships among the level of interleukin 25 (IL-25), the stage of liver fibrosis and the polarization of hepatic M2 macrophages in patients with non-alcoholic fatty liver disease (NAFLD). Methods A total of 36 patients with NAFLD and 20 control patients were enrolled. Fibrotouch, HE staining, and immunohistochemistry were used to evaluate the stage of liver fibrosis. Patients with NAFLD were classified into groups of mild liver fibrosis (F1) (20 cases) and significant liver fibrosis (≥ F2) (16 cases). The level of serum IL-25 in each group was detected by ELISA. Real-time fluorescent quantitative PCR was used to detect the hepatic mRNA expression levels of IL-25, collagen1 (Col1), α mooth muscle actin (α-SMA), macrophage mannose receptor 1 (CD206/MR1) and transglutaminase 2 (TGM2). Immunohistochemistry was used to detect the protein levels of IL-25, α-SMA, CD206 and TGM2. Results There was no significant difference in the level of serum IL-25 among groups. Compared with patients in the control group and the mild liver fibrosis group, patients with significant liver fibrosis showed reduced mRNA expression levels of IL-25, CD206, and TGM2 in addition to lower levels of hepatic IL-25 protein and less polarization of M2 macrophages. Conclusion Down-regulation of IL-25 is accompanied by a decrease in the number of the M2 macrophages with the progression of liver fibrosis in NAFLD patients.