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Galectin-3, a β-galactoside-binding animal lectin, is a multifunctional protein. Previous studies have suggested that galectin-3 may play an important role in inflammatory responses. Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a liver condition that may progress to end-stage liver disease and based on the known functions of galectin-3, it was hypothesized that galectin-3 might play a role in the development of NAFLD. Thus, this study investigated the role of galectin-3 in NAFLD by comparing galectin-3 knockout (gal3−/−) mice and wild-type (gal3+/+) mice. The livers of gal3−/− male mice at 6 months of age histologically displayed mild to severe fatty change. The liver weight per body weight ratio, serum alanine aminotransferase levels, liver triglyceride levels, and liver lipid peroxide in gal3−/− mice were significantly increased compared with those in gal3+/+ mice. Furthermore, the hepatic protein levels of advanced glycation end-products (AGE), receptor for AGE (RAGE), and peroxisome proliferator-activated receptor γ (PPARγ) were increased in gal3−/− mice relative to gal3+/+ mice. In conclusion, this study suggests that the absence of gal3 can cause clinico-pathological features in male mice similar to those of NAFLD.