ABSTRACT: Aim: The main objective of this study is to understand the mechanism of vancomycin resistance in a Streptomyces coelicolor disrupted mutant highly resistant to vancomycin. Materials & methods: Different techniques have been performed in the study including gene disruption, primer extension, antibiotic susceptibility tests, electron microscopy, confocal microscopy, cell wall analysis and microarrays. Results: During the phenotypical characterization of mutant strains affected in phosphate-regulated genes of unknown function, we found that the S. coelicolor SCO2594 disrupted mutant was highly resistant to vancomycin and had other phenotypic alterations such as antibiotic overproduction, impaired growth and reduction of phosphate cell wall content. Transcriptomic studies with this mutant indicated a relationship between vancomycin resistance and cell wall stress. Conclusion: We identified a S. coelicolor mutant highly resistant to vancomycin in both high and low phosphate media. In addition to Van proteins, others such as WhiB or SigE appear to be involved in this regulatory mechanism.