Abstract Oxytetracycline (OTC) is a widely used antibiotic, which is commercially produced by Streptomyces rimosus. The type II minimal polyketide synthases (minimal PKS) genes of the oxytetracycline biosynthesis cluster in S. rimosus, consisting of oxyA, oxyB and oxyC, are involved in catalyzing 19-C chain building by the condensation of eight malonyl-CoA groups to form the starting polyketide. This study aimed to investigate the effects of overexpression of the minimal PKS gene in a model S. rimosus strain (M4018) and in an industrial overproducer (SR16) by introduction of a second copy of the gene into the chromosome. Increased levels of oxyA, oxyB and oxyC gene transcription were monitored using reverse transcription quantitative real-time PCR. Overexpression of the minimal PKS gene elicited retardation of cell growth and a significant improvement in OTC production in corresponding mutants (approximately 51.2% and 32.9% in M4018 and SR16 mutants respectively). These data indicate that the minimal PKS plays an important role in carbon flux redirection from cell growth pathways to OTC biosynthesis pathways.