Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products. © 2014 American Chemical Society.