Hyperprolactinemia is prevalent in up to 16% of the infertile males. Although prolactin receptor (PRLR) is present on various testicular cells, the physiological role of this receptor in spermatogenesis remains elusive. The aim of this study is to delineate prolactin actions in rat testicular tissue. Serum prolactin, developmental expression of PRLR, signaling pathways associated, and gene transcription regulation in the testes were investigated. Serum prolactin and testicular PRLR expression was found to be significantly increased at pubertal and adult age as compared to pre-pubertal. Further, PRLR activated JAK2/STAT5 pathway, but not MAPK/ERK and PI3K/AKT pathway in the testicular cells. Gene expression profiling following prolactin treatment in seminiferous tubule culture resulted in a total of 692 differentially expressed genes, of which 405 were up-regulated and 287 were down-regulated. Enrichment map analysis showed that prolactin target genes are involved in processes like cell cycle, male reproduction, chromatin remodelling, and cytoskeletal organization. Novel targets genes of prolactin whose role in testes is unexplored were obtained and validated by qPCR. Additionally, ten genes involved in cell cycle process were also validated; six genes (Ccna1, Ccnb1, Ccnb2, Cdc25a, Cdc27, Plk1) were found to be significantly up-regulated, whereas four genes (Ccar2, Nudc, Tuba1c, Tubb2a) were found to be significantly down-regulated in testes after treatment with prolactin. Taken together, this study suggests a crucial role of prolactin in male reproduction and identified target genes regulated by prolactin in the testes.