Aim To observe the effect of TSB2 inhibiting the combination of heat shock protein 90 (HSP90) and endothelial nitric oxide synthase (eNOS) on the formation of atherosclerosis. Methods Human umbilical vein endothelial cells (HUVEC) were treated with TSB2 and the combination between HSP90 and eNOS was detected by co-immunoprecipitation. C57BL/6 mice and low density lipoprotein receptor knockout (LDLR－/－) mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks while injected with phosphate buffered saline (PBS) or TSB2 intraperitoneally. The mice were divided into four groups:C57BL/6＋ ND＋PBS group, LDLR－/－＋ND＋PBS group, LDLR－/－＋HFD＋PBS group, LDLR－/－＋HFD ＋TSB2 group. Then the aorta was isolated. The combination between HSP90 and eNOS in aorta was measured. The atherosclerotic plaque in aorta and aortic sinus were determined. The production of nitric oxide (NO) and superoxide anion (O₂·－) were also detected. At the same time, L-monomethyl-arginine (L-NMMA), a competitive substrate of L-arginine, was used to determine the production of NO, and L-nitroarginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used to determine the production of O₂·－. Results Compared with control group, the combination between HSP90 and eNOS was decreased by 41.06% (P＜0.05) in cultured HUVEC treated with TBS2. Compared with LDLR－/－＋HFD＋PBS group,the combination between HSP90 and eNOS in the mouse aortas was decreased by 40.95% (P＜0.05) in LDLR－/－ ＋HFD＋TSB2 group, and the production of O₂·－ was decreased by 63.73% (P＜0.05) (L-NAME significantly inhibited the production of O₂·－ in LDLR－/－＋HFD＋PBS group), while the production of NO had no significant change in the mouse aortic endothelial cells (L-NMMA inhibited NO production in all groups), and the formation of atherosclerotic lesions in aortas and aortic sinus were significantly decreased by 59.39% and 68.86% (P＜0.05) respectively in LDLR－/－＋HFD＋TSB2 group. Conclusion TSB2 can reduce the O₂·－ production of uncoupled eNOS in vascular endothelial cells by inhibiting the combination of HSP90 and eNOS in aortic endothelial cells, and finally inhibits the formation of atherosclerosis.