ABSTRACT: The Drosophila melanogaster male accessory gland (AG) is crucial for reproductive success, producing seminal fluid proteins that impact female post-mating behavior and physiology. To understand the regulatory networks governing accessory gland function, we conducted a bulk RNA sequencing (RNA-Seq) study to analyze the effects of overexpressing three key regulatory genes: dMyc, Yorkie (Yki), and the E2F-Dp heterodimer, compared to wild-type (WT) controls. We found upregulated and downregulated genes in each condition compared to controls, affecting immune response, cell cycle, stress response, and reproductive functions. In both Yki constitutively active, E2F+Dp and dMyc, E2F+DP conditions, several genes known to be involved in wound healing and tumorigenic models in flies were upregulated, including JNK signaling targets Ets21C, MMP1, puckered, and Jak/STAT signaling pathway ligands upd2 and upd3. Conversely, accessory gland-specific genes, such as many Accessory Gland Proteins (Acps), and genes involved in fertility, mating behavior, and sperm competition were downregulated. There was a strong overlap between the Yki constitutively active, E2F+Dp and dMyc, E2F+Dp gene expression signatures, converging on genes commonly misregulated in proliferative tumor models in various larval tissues, including imaginal discs and brains. This study provides insights into the molecular mechanisms by which key regulatory genes influence the function and integrity of the Drosophila male accessory gland.