Herein, two series of tryptanthrin derivatives with benzenesulfonamide substituents were designed and synthesized to discover novel anti-inflammatory agents. The anti-inflammatory activities of all derivatives were screened by evaluating their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. Among them, compound 8j exhibited the best NO inhibitory activity (IC50 = 1.25 ± 0.21 μM), with no obvious toxicity. Further evaluation showed that 8j could also significantly reduce the levels of pro-inflammatory cytokines interleukin-1β (IL-1β, IC50 = 8.48 ± 0.23μM) and tumor necrosis factor-α (TNF-α, IC50 = 11.53 ± 0.35 μM) and downregulate the LPS-induced expression of iNOS and COX-2. Reverse docking of 8j suggested p38α as the molecular target, which is a well-known crucial player in the p38 MAPK signaling pathway that controls the transcription of pro-inflammatory mediators. Cellular thermal shift assay showed that 8j efficiently stabilized p38α in LPS-treated RAW264.7 cells. Western blot showed that inflammatory response was inhibited by 8j through inhibiting the phosphorylation of p38α and MK2 in the p38 MAPK signaling pathway. Finally, In vivo studies showed that 8j could significantly ameliorate the degree of foot swelling and knee joint pathology in adjuvant-induced arthritis (AIA) rats and reduce levels of TNF-α and IL-1β in serum, achieving the effect of protecting synovial tissue and ameliorating arthritis. These findings suggested that 8j may be a promising compound for further development of anti-inflammatory agents.