A series of novel 4-substituted benzoxazolone derivatives was synthesized, characterized and evaluated as human soluble epoxide hydrolase (sEH) inhibitors and anti-inflammatory agents. Some compounds showed moderate sEH inhibitory activities in vitro, and two novel compounds, 3g and 4j, exhibited the highest activities with IC50 values of 1.72 and 1.07 μM, respectively. Structure-activity relationships (SARs) revealed that introduction of a lipophilic amino acid resulted in an obvious increase in the sEH inhibitory activity, especially for derivatives containing a phenyl (3d, IC(50) = 2.67 μM), pyrrolidine (3g, IC(50) = 1.72 μM), or sulfhydryl group (3e, IC(50)=3.02 μM). Several compounds (3a-3g) were tested in vivo using a xylene-induced ear edema mouse model. Three compounds (3d, 3f, and 3g) showed strong anti-inflammatory activities in vivo which were higher than that of Chlorzoxazone, a reference drug widely used in the clinic. Our investigation provided a novel type of sEH inhibitor and anti-inflammatory agent that may lead to the discovery of a potential candidate for clinical use.