Anti-folate agents are a class of drugs that exert their effects by inhibiting key enzymes in the folate metabolism pathway, thereby blocking cell proliferation and DNA synthesis. Family members include methotrexate (MTX), azathioprine (AZA), and others. These compounds structurally mimic folate, localizing in the cytoplasm and participating in one-carbon metabolism and nucleic acid synthesis. They impact various biological responses and signaling pathways, including cell cycle control and immune response. Specific expression patterns and gene mutations, such as DHFR gene mutations associated with MTX resistance, can affect their efficacy and toxicity. Therefore, personalized considerations are necessary in clinical applications.