Background Hemorrhage shock (HS) is characterized by decreased tissue oxygenation and organ damage due to severe blood loss. Acute lung injury (ALI) is a common complication of HS. Protein tyrosine phosphatase receptor type O (PTPRO) is abnormally up-regulated in the rat lungs after trauma/HS. Methods To elucidate the regulatory mechanism of PTPRO in lung inflammation following HS, we established a rat model of HS via withdrawing blood by a catheter inserted into the femoral artery followed by resuscitation. The rats were infected with lentiviral encoding PTPRO short hairpin RNA (shRNA) by intratracheal instillation for PTPRO knockdown. Results PTPRO was significantly up-regulated in rat lungs after HS. PTPRO knockdown enhanced epithelial integrity and reduced capillary leakage by up-regulating tight junction proteins zonula occludens-1 (ZO-1) and occludin (OCC). Besides, decreased myeloperoxidase expression and activity in the lungs indicated that HS-induced neutrophil infiltration into the lungs was mitigated by PTPRO knockdown. Meanwhile, expression of inflammatory cytokines/chemokines TNF-α, IL-6, MIP-2, MCP-2, and KC in the lungs or bronchoalveolar lavage fluid was regressed after PTPRO knockdown. The nuclear factor kappa B (NF-κB) pathway was related to inflammation in organ injury. PTPRO down-regulation had an inhibitory effect on the NF-κB pathway activation by suppressing the phosphorylation of NF-κB and its translocation from the cytoplasm into the nucleus in HS. Conclusion Taken together, we demonstrated that PTPRO knockdown may contribute to attenuating inflammation in HS-induced acute lung injury via inhibiting NF-κB pathway activation.