Daptomycin is a calcium-dependent cyclic lipopeptide antibiotic. It is one of only two new structural classes of antibiotics that have been approved for clinical use over the last 40 years. It is used to treat serious infections caused by Gram-positive bacteria. Although daptomycin has been used in the clinic since 2003, clinical resistance to daptomycin is not yet widespread. However, reports of clinical isolates that are resistant to daptomycin have been increasing in recent years, which is a cause for concern since daptomycin is often used to treat serious infections that are resistant to other antibiotics. Structural variation of antibiotics is a strategy that has often been used to overcome bacterial resistance. To pursue such a strategy with daptomycin, knowledge of its mechanism of action and methods for preparing daptomycin analogues, and the development of daptomycin structure–activity relationships (SARs) are necessary. This article, which is based on the 2023 Bernard Belleau Award Lecture, provides an overview of some of the key investigations that were undertaken by the Taylor group on daptomycin, including mechanism of action studies, the total synthesis of daptomycin, daptomycin SARs, and characterization of the interaction of daptomycin with phosphatidylglycerol, its primary target.