Cytokines-mediated immunity is essential for the pathological development of rheumatoid arthritis (RA). Inhibition of signaling has suggested a potential remedial approach to RA. G protein-coupled receptor 4 (GPR4) has been proven to possess a broad range of physiological functions, but its function in synovial mast cells and RA is less reported. In this study, the protective effects of NE 52-QQ57, a GPR4 antagonist, against interleukin (IL)-33-challenged inflammatory response in activated synovial mast cells were investigated. We report that IL-33 amplified GPR4 expression in HMC-1 mast cells. The GPR4 antagonist NE 52-QQ57 alleviated IL-33-caused secretions of IL-17, interferon-γ, and tumor necrosis factor-α in HMC-1 mast cells. Furthermore, we note that NE 52-QQ57 reduced IL-33-induced expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Also, NE 52-QQ57 inhibited cyclooxygenase 2 and prostaglandin E2 expression in IL-33-challenged cells. Also, NE 52-QQ57 ameliorated IL-33-induced oxidative stress by reducing mitochondrial reactive oxygen species and 4-hydroxynonenal. Mechanistically, NE 52-QQ57 mitigated IL-33-induced activation of the p38/nuclear factor-κB signaling pathway. We conclude that targeting GPR4 might be a promising strategy for RA treatment.