Cancer is a life-threatening destructive disease. In the past several decades, the incidence of cancer has been dramatically increased mostly due to lifestyle changes. Chemotherapy plays an important role in the treatment of cancer, but the development of resistance against chemotherapeutic agents, the side effects, and non-specific toxicity threatens the efficiency of anticancer agents. Accordingly, it is important to develop novel anticancer drugs. Beyond the classical antibacterial activity, macrolides also demonstrated potential effects against both drug-sensitive and drug-resistant cancers through modulating diverse targets and signaling pathways, so rational design of macrolides may generate valuable therapeutic interventions for the treatment of cancers. The purpose of the present review article is to outline the current developments in macrolides with an emphasis on anticancer activity, structure-activity relationships, and mechanisms of action to lay the path for the development of novel macrolide anticancer candidates.