Interleukin (IL)-32 is a multifunctional cytokine involved in inflammation regulation, immune response, and tumor biology. The IL-32 gene consists of eight exons and produces nine different isoforms through alternative splicing. First identified in activated T cells and natural killer cells, the expression of IL-32 is increased by various stimuli, including microorganisms, mitogens, and inflammatory cytokines. IL-32 is widely expressed in various human tissues, including the spleen, thymus, lungs, liver, and intestines. Its expression is particularly prominent in immune cells, where it plays a key role in regulating cell growth, metabolism, and immune responses, and is associated with various inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, infectious diseases, and metabolic syndrome) and malignant tumors (e.g., esophageal cancer, hepatocellular carcinoma, gastric cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and lymphoma). IL-32 has been reported to promote or inhibit inflammatory responses and tumor development by regulating major signaling pathways such as nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STAT) 3, and mitogen-activated protein kinase (MAPK), depending on its isoform and cellular context. Nevertheless, a comprehensive understanding of the isoform-specific roles and molecular mechanisms of IL-32 in cancer and inflammatory diseases remains incomplete. Therefore, in this review, we aimed to summarize the current literature on the function of IL-32 in the development of inflammatory diseases and cancer, and discuss potential therapeutic strategies targeting IL-32 through emerging big data analysis.