<p>Cytotoxic CD4⁺ T lymphocytes are a unique subset of CD4⁺ T cells characterized by their cytokine secretion and cytolytic activity. Unlike traditional CD4⁺ helper T cells, cytotoxic CD4⁺ T lymphocytes exhibit similar cytotoxicity to that of CD8⁺ T cells, revealing unexpected plasticity in CD4⁺ T-cell functions. This flexibility suggests that CD4⁺ T cells can transform and acquire effector functions beyond their conventional functions, emphasizing their importance in various immune responses. Despite the identification of cytotoxic CD4⁺ T lymphocytes decades ago, recent advancements have broadened our understanding of their phenotypic diversity, transcriptional regulation, differentiation pathways, and functional roles. Cytotoxic CD4⁺ T lymphocytes play pivotal roles across a broad spectrum of diseases, including cancer, infectious diseases, autoimmune disorders, and cardiovascular diseases. They typically mediate strong inflammatory responses and kill target cells through cytotoxicity, playing a crucial role in maintaining immune homeostasis. In this review, we synthesize current findings on cytotoxic CD4⁺ T lymphocytes, emphasizing their origin, biomarkers, regulatory molecules, and biological functions. Additionally, we focus on their pathological roles in the progression of various diseases and examine how cytotoxic CD4⁺ T lymphocytes contribute to disease development and progression. We also provide a comprehensive summary of therapeutic strategies targeting cytotoxic CD4⁺ T cells and review the associated clinical trial data, aiming to propose new strategies for disease management through the targeting of cytotoxic CD4⁺ T lymphocytes.</p>