Endogenous AICAR (acadesine) demonstrates significant therapeutic potential as a phase Ⅲ clinical agent for the treatment of adverse cardiovascular reactions to coronary artery bypass grafting and as a phase Ⅰ/Ⅱ clinical agent for chronic lymphocytic leukaemia. However, its biosynthetic mechanism remains poorly defined. Our previous study demonstrated that AICAR was significantly enriched in the Fusarium solani mutant veA^OE14, which overexpressed the global regulator VeA. We found that phosphoribosyl pyrophosphate synthase 2, viz., PRPS2 (PrsA) is regulated by VeA and involved in AICAR synthesis. Deletion of PRPS2 resulted in a significant 17% (μg/mg) reduction in AICAR production in the strain (p < 0.05), whereas its overexpression led to a statistically significant 1.2-fold increase in AICAR content (p < 0.01). Furthermore, we characterised the transcription factor MtfA, which inhibits the expression of PRPS2 gene and is negatively regulated by VeA. Further validation by yeast one-hybrid assay showed that MtfA directly regulated the PRPS2 gene, while AICAR was significantly increased in the mtfA deletion mutant. The above results indicate that the global regulator VeA negatively regulates the transcription factor MtfA, which in turn targets negatively regulating transcriptional levels of PRPS2 to mediate AICAR biosynthesis in F. solani.