Objective To investigate the effect of butorphanol on lipopolysaccharide-induced chondrocyte injury by regulating the stromal cell-derived factor-1α (SDF-1α)/C-X-C chemokine receptor 4 (CXCR4) pathway. Methods Human C28/I2 chondrocytes were cultured in vitro and assigned to the following groups: control (normal culture), model (100 μmol/L lipopolysaccharide), model+low-dose butorphanol (100 μmol/L lipopolysaccharide+1 μmol/L butorphanol), model+medium-dose butorphanol (100 μmol/L lipopolysaccharide+2 μmol/L butorphanol), model+high-dose butorphanol (100 μmol/L lipopolysaccharide+4 μmol/L butorphanol), and model+high-dose butorphanol+NUCC-390 (100 μmol/L lipopolysaccharide+4 μmol/L butorphanol+500 nmol/L CXCR4 agonist NUCC-390). Cell viability, interleukin (IL) -6 and tumor necrosis factor-α (TNF-α) levels, apoptosis, and SDF1α/CXCR4 pathway-related proteins were evaluated by MTT assay, enzyme-linked immunosorbent assay, flow cytometry, and Western blot, respectively. Results Chondrocyte survival rate and Bcl-2 protein expression were decreased while TNF-α, IL-6, apoptosis rate, Bax, Cleaved caspase-3, SDF-1α, and CXCR4 proteins were increased in the model group compared with the control group (P<0.05). The above indicators were improved in the model+low-, medium-, and high-dose butorphanol groups compared with the model group, while the result for the model+high-dose butorphanol+NUCC-390 group were opposite to those of the model+high-dose butorphanol group. Conclusions Butorphanol may improve lipopolysaccharide-induced chondrocyte injury induced by inhibiting the SDF-1α/CXCR4 signaling pathway.