Background: Atherosclerosis, the underlying cause of the majority of cardiovascular diseases, is a lipid-driven, inflammatory disease of the large arteries. Atherosclerotic cardiovascular disease (ASCVD) threatens human lives due to high morbidity and mortality. Many studies have demonstrated that atherosclerosis is accelerated via activation of the NLRP3 inflammasome. The NLRP3 inflammasome plays a critical role in the development of vascular inflammation and atherosclerosis. In atherosclerotic plaques, excessive generation of reactive oxygen species (ROS) activates the NLRP3 inflammasome. 13-Methylberberine (13-MB) is a newly synthesized compound used in traditional Chinese medicine that has outstanding antibacterial, antitumor, and antiobesity activities, especially anti-inflammatory activity. However, the role of 13-MB in atherosclerosis needs to be explored. Methods: CCK-8 assays and flow cytometry were conducted to determine the cell viability and apoptotic profiles of human umbilical vein endothelial cells (HUVECs) treated with 13-MB. Carboxy-DCFH-DA and JC-10 assays were used to measure ROS and determine mitochondrial membrane potential. Western blot analysis was performed to investigate proteins that are associated with the NLRP3 inflammasome and autophagy. ELISA was used to detect and quantify inflammatory cytokines related to the NLRP3 inflammasome. Transfection and confocal microscopy were conducted to observe autophagy. Results: Pretreatment with 13-MB markedly reduced cytotoxicity and apoptosis, as well as intracellular ROS production, in HO-induced HUVECs. Moreover, 13-MB showed a protective effect in maintaining mitochondrial membrane potential. 13-MB also suppressed NLRP3 inflammasome activation and promoted autophagy induction in HUVECs. Conclusion: 13-MB exerts cytoprotective effects in an HO-induced cell injury model by inhibiting NLRP3 inflammasome activation via autophagy induction in HUVECs. These anti-inflammatory and autophagy induction activities may provide valuable evidence for further investigating the potential role of 13-MB in atherosclerosis.