Previously, we noted that SLIT3, slit guidance ligand 3, had an osteoprotective role with bone formation stimulation and bone resorption suppression. Additionally, we found that global Slit3 KO mice had smaller long bone. Skeletal staining showed short mineralized length in the newborn KO mice and wide hypertrophic chondrocyte area in the embryo KO mice, suggesting delayed chondrocyte maturation. The recombinant SLIT3 did not cause any change in proliferation of ATDC5 cells, but stimulated expressions of chondrocyte differentiation markers, such as COL2A1, SOX9, COL10A1, VEGF, and MMP13 in the cells. SLIT3 suppressed β-catenin activity in the cells, and activation of Wnt/β-catenin signaling by lithium chloride attenuated the SLIT3-stimulated differentiation markers. ATDC5 cells expressed only ROBO2 among their 4 isotypes, and the Robo2 knock-down with its siRNA reversed the SLIT3-stimulated differentiated markers in chondrocytes. Taken together, these indicate that SLIT3/ROBO2 promotes chondrocyte maturation via the inhibition of β-catenin signaling.