Objective To explore the effect of tribbles homologue 3 (TRB3) on spinal astrocytes injury induced by the oxygen-glucose deprivation/reoxygenation (OGD/R) in rats. Methods The spinal astrocytes of newborn SD rats were isolated and cultured. Cells were divided into control group (without treatment) and OGD/R group (cells were exposed to OGD/R). The mRNA and protein expression levels of TRB3 were detected by Real-time PCR and Western blotting. Cells were divided into control group, Scramble group (infected with scramble virus) and shTRB3 group (infected with TRB3 shRNA lentivirus), and the mRNA and protein expression levels of TRB3 were detected by Real-time PCR and Western blotting to confirmed knockdown efficiency.Cells were divided into control group, OGD/R group, Scramble group, shTRB3 group, OGD/R+Scramble group (cells were infected with scramble virus, and then treated with OGD/R) and OGD/R+shTRB3 group (cells were infected with TRB3 shRNA lentivirus,and then treated with OGD/R). The cell apoptosis, cell viability and lactic dehydrogenase (LDH) leakage rate were detected by flow cytometry, CCK-8 assay, and LDH detection kit, respectively. Malondialdehyde (MDA) content was detected by the TBA method and the activity of superoxide dismutase (SOD) was tested by the xanthine oxidase method. The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by ELISA. The protein expression of nuclear factor kappa-B (NF-κB)p65 was measured by Western blotting. Cells in OGD/R and OGD/R+shTRB3 were also treated with NF-κB p65 agonist betulinic acid (BA, 20 μmol/L), and cell apoptosis was detected by flow cytometry. Results Real-time PCR and Western blotting results showed that the mRNA (2.15±0.12 vs. 1.00±0.05) and protein (2.10±0.16 vs. 1.00±0.08) expression levels of TRB3 in spinal astrocytes were upregulated after OGD/R (P<0.05). Compared with control group, TRB3 shRNA lentivirus infection significantly decreased mRNA (0.30±0.07 vs. 1.00±0.10) and protein (0.30±0.04 vs. 1.00±0.06) expression levels of TRB3 (P<0.05).Compared with control group, OGD/R group and OGD/R+Scramble group showed significantly decreased cell viability, SOD activity and nuclear NF-κB p65 level, and increased LDH, MDA, TNF-α, IL-1β levels and cell apoptosis rate (P<0.05), indicated that TRB3 shRNA lentivirus infection is successful. Compared with OGD/R group and OGD/R+Scramble group, TRB3 silencing significantly inhibited OGD/R induced decreased cell viability, SOD activity and nuclear NF-κB p65 level (P<0.05). And the increase of LDH, MDA, TNF-α, IL-1β and apoptosis induced by OGD/R was significantly inhibited by TRB3 silencing (P<0.05).Compared with OGD/R group, NF-κB p65 activator BA treatment could significantly increase cell apoptosis rate (36.15%±0.87%vs. 24.70%±1.05%, P<0.05), and it also could reverse the effect of TRB3 silencing on OGD/R induced cell apoptosis rate(22.00%±1.04% vs. 13.91%±1.20%, P<0.05). Conclusion TRB3 silencing inhibits OGD/R-induced rat spinal astrocyte injury,which may be mediated by blocking the NF-κB pathway.