ABSTRACT Brown eye spot, caused by Cercospora coffeicola , causes significant losses in both quality and quantity of coffee production. As many Cercospora spp. produce the photoactivated toxin cercosporin, this study aimed to determine the role of cercosporin in C. coffeicola pathogenesis by creating disruption mutants unable to produce the toxin. Six C. coffeicola isolates from Brazilian fields, representing organic and conventional production systems in the Minas Gerais state, were evaluated for their ability to produce cercosporin in vitro . Toxin production varied among isolates, ranging from 3.5 – 25.3 µM/ 5 mm mycelial plug; production was undetectable in one isolate. The C. coffeicola homolog of the polyketide synthase gene ( CTB1 ) involved in cercosporin production was amplified using a degenerate primer strategy. The 7044 nt ccCTB1 gene sequence was 90.3% identical to the cnCTB1 gene in Cercospora nicotianae and encoded a putative protein of 2196 amino acids with 98.2% similarity and 97.5% identity to its counterpart in C. nicotianae . Transformation of two isolates of C. coffeicola with a CTB1 disruption construct resulted in the recovery of six ctb1 disruption mutants. All of the ctb1 disruptants were deficient in cercosporin production. Disruption mutants did not differ significantly from the wild type for either growth or sporulation, but were significantly altered in virulence on coffee. As compared to wild type, time to lesion development was significantly increased and numbers of lesions were significantly decreased in coffee plants inoculated with ctb1 disruption mutants. These results show that cercosporin toxin is a virulence factor for C. coffeicola infection of coffee.