The galbonolide antibiotics are non-glycosylated heptaketide 14-membered macrolides. These antibiotics exhibit broad-spectrum fungicidal activities, including against the human pathogen Cryptococcus neoformans. Previously, galbonolides B and E were isolated from the marine actinomycete Streptomyces sp. LZ35. By bioinformatics analysis, the putative galbonolide biosynthetic gene cluster, gbn, was identified in the genome of strain LZ35. In order to verify that the core genes (gbnA–E) are sufficient for synthesizing the basic structure of galbonolide as previously proposed, we performed the heterologous expression of gbnA–E in a “clean background” host Streptomyces coelicolor ZM12, in which all the native polyketide synthase genes have been deleted. As expected, the production of galbonolide B (1) was detected in the transformant. To the best of our knowledge, this is the first report that demonstrates the essential role of gbnA–E in the biosynthesis of galbonolides by heterologous expression. This heterologous expression system would be helpful to generate novel galbonolide derivatives by co-overexpression of unusual biosynthesis extender units.