Non-alcoholic fatty liver disease (NAFLD) is a complex disease ranging from steatosis to non-alcoholic steatohepatitis (NASH). Galectin-3 (Gal-3), which is a β-galactoside binding protein, has been associated with liver fibrosis, but its role in NAFLD remains elusive. We investigated the expression of Gal-3 in liver resident cells and its potential association with liver damage in 40 children with biopsy-proven NAFLD. We found that several liver cells expressed Gal-3. The number of total Gal-3 positive cells decreased with the severity of disease and the cells were correlated with the presence of steatosis and the diagnosis of NASH. CD68 macrophages expressed Gal-3 but the number CD68/Gal-3 positive cells was significantly reduced in patients diagnosed with steatosis and NASH. Triple CD68/CD206/Gal-3, which represented the subpopulation of M2 macrophages, were mainly present in patients without NASH, and clearly reduced in patients with steatosis and NASH. On the contrary, the number of α-smooth muscle actin (SMA)/Gal-3 positive cells increased with the severity of fibrosis in children with NAFLD. Our data demonstrated that the number of Gal-3 positive cells was associated with tissue damage in different ways, which suggests a dual role of this protein in the pathogenesis of pediatric NAFLD, even if the role of Gal-3 deserves further studies.