Abstract: Lymphocyte activation gene 3 (LAG-3) is an important inhibitory receptor on T cells, which plays a crucial role in tumor immune evasion. LAG-3 is primarily expressed on activated T cells, natural killer (NK) cells and B cells, et al. By binding to its ligands, LAG-3 inhibits T cell proliferation, activation, and effector functions. LAG-3 has emerged as the third immune checkpoint protein (ICP) used in clinical practice, following programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Currently, there has been at least 20 LAG-3-targeted drugs undergoing clinical trials. This article mainly reviews the structure, expression regulation, ligands, co-expressed ICP of LAG-3, as well as its application in tumor immunotherapy, and discusses the current challenges of targeting LAG-3 research.