Background: Upadacitinib (UPA) is a selective tyrosine kinase 1 (JAK-1) inhibitor, which has been applied to treat atopic dermatitis, psoriatic arthritis, and ulcerative colitis in clinic. Whether it can treat sepsis remains unclear. Here, we investigate the effect of UPA on lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-induced inflammatory responses in vitro and in vivo. Methods: In vitro, LPS-treated RAW264.7 cell line, LPS-treated TLR4 knock-out (TLR4-/-) RAW264.7 cell line, and LPS-treated NLRP3 knock-out (NLRP3-/-) RAW264.7 cell line were used. In vivo, CLP-treated mice and CLP-treated TLR4-/- mice were used. Proteomics was used to screen inflammation-related differential proteins in RAW264.7 cells after treatments. After that, Western blotting was used to investigate the potential mechanism. Results: In vitro, UPA significantly inhibited TLR4/NF-kappa B and JAK/STAT pathway in the LPS-treated RAW264.7 cells. In addition, UPA reduced protein expressions of NF-kappa B and its downstream inflammatory cytokines such as TNF-alpha, IL-1(3 in the LPS-treated TLR4-/- RAW264.7 cells. In vivo, UPA markedly protected the sepsis mice, decreased intestinal injuries, reduced bacterial load, and downregulated the TLR4/NF-kappa B and JAK/STAT pathways-related proteins in macrophages isolated from peritoneal lavage fluids (PLFs) of the sepsis mice. In fact, UPA still exerted the protective effect in the CLP-treated TLR4-/- mice. The proteomics revealed that NOD-like receptor (NLR) signaling was one of the most significantly affected pathways between the LPS-treated and UPAtreated. Although UPA significantly reduced NLRP3 and IL-1(3 protein expressions in the LPS-treated RAW264.7 cells, its antiinflammatory effect was not significantly abolished in the LPS-treated NLRP3-/- RAW264.7 cells. Conclusion: Taken together, UPA inhibits the LPS- and CLP-induced inflammatory responses in vitro and in vivo, which is associated with the inhibition of NF-kappa B and its downstream cytokines.