Lipopolysaccharide (LPS) was used to induce mouse mononuclear macrophages (RAW 264.7 cells) to establish the inflammation model for investigating the effect and mechanism of Mailuoning oral liquid on arteriosclerosis occlusion (ASO) in vitro. RAW264.7 cells viability was measured by MTT assay. NO concentration was determined by Griess. mRNA levels and protein expressions of NFAT5/NLRP3 signaling pathway were detected by Q-PCR and Western blot. The relationship between NFAT5 and NLRP3 was explored by cellular transfection of NFAT5-siRNA combined with Western blot. Nuclear translocation of NFAT5 was detected by immunofluorescence. The results showed that Mailuoning oral liquid decreased the NO release induced by LPS in RAW264.7 cells. The mRNA levels of NFAT5, NLRP3, caspase1, IL-18 and MMP9, the protein expressions of NFAT5, NLRP3, cleaved-caspase1 (p20) and the phosphorylation of NF-κB-P65 were decreased after administration of Mailuoning oral liquid. NFAT5-siRNA significantly reversed the increase in protein expressions of NLRP3 induced by LPS in RAW264.7 cells. Both Mailuoning oral liquid and KRN2 (NFAT5 inhibitor) could inhibit the expressions and nuclear translocation of NFAT5. In conclusion, Mailuoning oral liquid exert significant anti-inflammatory effects in vitro by inhibiting the NFAT5/NLRP3 signaling pathway, and NFAT5 might be involved in regulating the expressions of NLRP3.