Aim To study the expression and biological significance of microRNA-499 (miR-499) and high mobility group protein 1 (HMGB1) in carotid atherosclerotic plaque. Methods Patients with carotid atherosclerosis diagnosed in Sanmenxia Central Hospital of Henan Province from January 2018 to December 2020 were enrolled in the carotid atherosclerosis group, and healthy people were enrolled in the control group. The expression level of miR-499 in peripheral blood and the content of HMGB1 in serum were detected. Apolipoprotein E (ApoE) knockout mice were divided into groups and fed with high cholesterol diet to establish carotid atherosclerotic plaque model. miR-NC, miR-499, NC lentivirus (lenti-NC) and HMGB1 lentivirus (lenti-HMGB1) were given by tail vein injection. Then the pathological changes, the expression levels of miR-499 and HMGB1 in carotid atherosclerotic plaque were determined. Human umbilical vein endothelial cells (HUVEC) were cultured and double luciferase reporter gene was used to verify the targeted binding of miR-499 and HMGB1. Results The expression level of miR-499 in peripheral blood of carotid atherosclerosis group was lower than that of control group, and the content of HMGB1 in serum was higher than that of control group, and the expression level of miR-499 was negatively correlated with the content of HMGB1 in serum (P＜0.05). The expression level of miR-499 in carotid atherosclerotic plaque of model group was lower than that of control group, and the expression level of HMGB1 was higher than that of control group; the pathological changes of carotid atherosclerotic plaque in miR-499+model group were better than those in miR-NC+model group, the expression level of miR-499 in carotid atherosclerotic plaque was higher than that of miR-NC+model group, the expression level of HMGB1 was lower than that of miR-NC+model group. The pathological changes of carotid atherosclerotic plaque in lenti-HMGB1+miR-499+model group were more severe than those in lenti-NC+miR-499+model group, and the expression level of HMGB1 was higher than that in lenti-NC+miR-499+model group. The fluorescence value of wild-type HMGB1 reporter gene in miR-499 group was significantly lower than that in miR-NC group (P＜0.05). Conclusion miR-499 targeting HMGB1 is involved in the formation of carotid atherosclerotic plaque.