Endothelial dysfunction is a significant factor in the pathogenesis of various diseases. In pathological states, endothelial cells (ECs) undergo activation, resulting in dysfunction characterized by the stimulation of inflammatory responses, oxidative stress, cell proliferation, blood coagulation, and vascular adhesions. Interleukin-35 (IL-35), a novel member of the IL-12 family, is primarily secreted by regulatory T cells (Tregs) and regulatory B cells (Bregs). The role of IL-35 in immunomodulation, antioxidative stress, resistance to apoptosis, control of EC activation, adhesion, and angiogenesis in ECs remains incompletely understood, as the specific mechanisms of IL-35 action and its regulation have yet to be fully elucidated. Therefore, this systematic review aims to comprehensively investigate the impact of IL-35 on ECs and their physiological roles in a range of conditions, including cardiovascular diseases, tumors, sepsis, and rheumatoid arthritis (RA), with the objective of elucidating the potential of IL-35 as a therapeutic target for these ailments.