Melatonin is a known modulator of follicle development; it acts through several molecular cascades via binding to its two specific receptors MT1 and MT2. Even though it is believed that melatonin can modulate granulosa cell (GC) functions, there is still limited knowledge of how it can act in human GC through MT1 and MT2 and which one is more implicated in the effects of melatonin on the metabolic processes in the dominant follicle. To better characterize the roles of these receptors on the effects of melatonin on follicular development, human granulosa-like tumor cells (KGN) were treated with specific melatonin receptor agonists and antagonists, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p ≤ 0.05) for each treatment, lists of the principal differentially expressed genes (DEGs) are generated. Analysis of major upstream regulators suggested that the MT1 receptor may be involved in the melatonin antiproliferative effect by reprogramming the metabolism of human GC by activating the PKB signaling pathway. Our data suggest that melatonin may act complementary through both MT1 and MT2 receptors to modulate human GC steroidogenesis, proliferation, and differentiation. However, MT2 receptors may be the ones implicated in transducing the effects of melatonin on the prevention of GC luteinization and follicle atresia at the antral follicular stage through stimulating the PKA pathway.