Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a dual mode of action. This study sought to understand the mechanisms of decreased telavancin susceptibility in a laboratory-derived Staphlococcus aureus mutant Tlv(DS)MED1952. There were extensive changes in the transcriptome of Tlv(DS)MED1952 compared to the susceptible parent strain MED1951. Genes upregulated included cofactor biosynthesis genes, cell wall-related genes, fatty acid biosynthesis genes, and stress genes. Downregulated genes included lysine operon biosynthesis genes and lrgB, which are induced by telavancin in susceptible strains, agr and kdpDE genes, various cell surface protein genes, phenol-soluble modulin genes, several protease genes, and genes involved in anaerobic metabolism. The decreased susceptibility mutant had somewhat thicker cell walls and a decreased autolytic activity that may be related to decreased proteolytic peptidoglycan hydrolase processing. Membrane fatty acid changes correlated with increased membrane fluidity were observed. It seems likely that there are multiple genetic changes associated with the development of decreased telavancin susceptibility. The Tlv(DS) mutant showed some similar features to vancomycin-intermediate S. aureus and decreased daptomycin susceptibility strains, but also exhibited its own unique features.