Despite the effectiveness and tolerability of antiretroviral therapy (ART) in treating HIV infection, integrase strand transfer inhibitors, particularly dolutegravir (DTG)-based ART, have been associated with various brain complications. Our research group has previously reported that DTG disrupts the blood-brain barrier (BBB) by inducing endoplasmic reticulum (ER) stress. To further understand DTG-associated toxicity, this follow-up study assessed autophagy dysfunction, a critical process that is closely linked to ER stress, using primary cultures of mouse brain microvascular endothelial cells as a robust BBB rodent model. We demonstrated that DTG exposure at therapeutically relevant concentrations significantly increased Sqstm/p62, LC3B-I and LC3B-II protein expression, and downregulated autophagy-related genes Ulk1 and Atg14, suggesting an autophagy inhibition as a result of DTG treatment. This study provides new insights into the toxicological mechanisms of DTG and pathogenesis of DTG-associated brain complications.