Piroxicam (PX) is potent and widely used for the treatment of inflammatory conditions. To develop active PX analogues with improved anti-inflammatory activity and minimized adverse effects, a series of PX analogues were designed and synthesized for enhancing anti-inflammatory activity. Among these derivatives, compound 12 exhibited superior NO-inhibitory ability with minimal cytotoxic effect in LPS-induced RAW 264.7 macrophage cells. Mechanistically, network pharmacology analysis and western blot assays revealed that the protective effect of compound 12 was attributed to the regulation of MEK/ERK signaling pathway inhibition in RAW 264.7 macrophages. Molecule docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay indicated the direct interaction between compound 12 and MEK protein. Moreover, compound 12 demonstrated great inhibition of LPS-induced MEK activation and subsequent demonstrated great inhibition of LPS-induced NF-kappaB activation than compound PX. Furthermore, compared to compound PX, 10 mg/kg dose of compound 12 demonstrated improved mitigation of phenotypes in 2 % dextran sulfate sodium (DSS)-induced mouse colitis model. The inhibitory effect of compound 12 on MEK/ERK signaling pathway in colonic tissues was superior to that of compound PX. Overall, our study highlights compound 12 as a promising novel piroxicam analogue candidate for anti-inflammatory therapy, offering potential advancements in human inflammatory bowel disease (IBD) treatment.