Apicidin is a cyclic tetrapeptide produced by certain isolates of Fusarium semitectum and has been shown to inhibit Apicomplexan histone deacetylase. An apicidin-producing strain (KCTC16676) of the filamentous fungus was mutated using an Agrobacterium tumefaciens-mediated transformation, resulting in 24 apicidin-deficient mutants. Three of the mutants had a T-DNA insertion in a gene that encodes a non-ribosomal peptide synthetase (NRPS). Results of sequence, expression, and gene deletion analyses defined an apicidin biosynthetic gene cluster, and the NRPS gene was named as apicidin synthetase gene 1 (APS1). A 63 kb region surrounding APS1 was sequenced and analysis revealed the presence of 19 genes. All of the genes including APS1 were individually deleted to determine their roles in apicidin biosynthesis. Chemical analyses of the mutant strains showed that eight genes are required for apicidin production and were used to propose an apicidin biosynthetic pathway. The apicidin analogues apicidin E, apicidin D2 and apicidin B were identified from chemical analysis of the mutants. The cluster gene APS2, a putative transcription factor, was shown to regulate expression of the genes in the cluster and overexpression of APS2 increased apicidin production. This study establishes the apicidin biosynthetic pathway and provides new opportunities to improve the production of apicidin and produce new analogues.