Objective To investigate the protective effect and mechanism of recombinant human interleukin 35(rhIL-35) on coronary artery injury in Kawasaki disease (KD). Methods Human coronary artery endothelial cells (HCAECs) were cultured in vitro to establish KD vascular model. Tumor necrosis factor α(TNF-α) and the serum of KD patients stimulated HCAECs were used to mimic the local inflammatory lesions of KD. The cells were divided into control group, TNF-α and KD serum stimulation group, (25, 50) ng/mL rhIL-35 treatment group. Cell proliferation was detected by CCK-8 assay; mRNA levels of IL-1β, IL-6, IL-17A and zonula occludens-1(ZO-1) of HCAECs were detected by real-time quantitative PCR; IL-35 expression in plasma and IL-1β, IL-6 and IL-17A content in HCAEC supernatant were tested by ELISA; Western blot was performed to detect the expression of nuclear factor κB p65 (NF-κB p65) and ZO-1. Results TNF-α and KD serum inhibited the proliferation of HCAECs, while rhIL-35 significantly reversed the above effects. RhIL-35 significantly down-regulated the expression of IL-1β, IL-6 and IL-17A after preconditioning HCAECs. Compared with TNF-α and KD serum stimulation group, rhIL-35 pretreated cells could significantly increase ZO-1 protein expression and inhibit NF-κB p65 expression. Conclusions rhIL-35 can alleviate the damage of KD coronary artery endothelial cells by inhibiting NF-κB pathway.