Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a β-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)D. The results showed that 1α,25(OH)D upregulated the expression of mRNA and receptor activator of nuclear-factor κB ligand () expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (, , , and ) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of by small-interfering RNA (siRNA) in BMSCs downregulated the expression of mRNA and attenuated the effects of 1α,25(OH)D on osteoclastogenesis and bone resorption. Conversely, overexpression of in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)D.