Abstract Aging is a multifaceted biological process marked by the decline in both mitotic and postmitotic cellular function, often central to the development of age-related diseases. In the pursuit of slowing or even reversing the aging process, a prominent strategy of significant interest is calorie restriction (CR), also known as dietary restriction, and the potential influence of a drug called rapamycin (RM). Both CR and RM have demonstrated the capacity to extend healthspan and lifespan across a diverse array of species, including yeast, worms, flies, and mice. Nevertheless, their individual and combined effects on mitotic and postmitotic cells, as well as their comparative analysis, remain areas that demand a thorough investigation. In this study, we employ RNA-sequencing methodologies to comprehensively analyze the impact of CR, RM, and their combination (CR + RM) on gene expression in yeast cells. Our analysis uncovers distinctive, overlapping, and even contrasting patterns of gene regulation, illuminating the unique and shared effects of CR and RM. Furthermore, the transcriptional synergistic interaction of CR + RM is validated in extending the lifespan of both yeast and human cells.