Previous studies have strongly recommended that KISS-1 metastasis suppressor (KISS1) plays an essential gatekeeper of the initiation of reproductive maturation in mammals. However, KISS1 has been recently reported to highly express in ovarian granulosa cells (GCs). But the biological functionalities of KISS1 on cell apoptosis, cell cycle, and synthesis of estradiol-17β (E2) have not been explored in GCs. In this study, using porcine GCs as a cellular model, the overexpression plasmid of KISS1 was built to explore the biological effects of KISS1 on the PI3K signaling pathway, estrogen signaling pathway, cell apoptosis, cell cycle, and E2 secretion. We found that mRNA of KISS1 highly expressed in the ovary and significantly increased from immature to mature follicles in gilts. Overexpression of KISS1 could significantly increase the mRNA expression of PIK3CG, PIK3C1, and PDK1, and significantly decreased the mRNA levels of FOXO3, TSC2, and BAD of PI3K signaling pathway. Furthermore, results of the flow cytometry showed that overexpression of KISS1 significantly inhibited the apoptosis of GCs and decreased the percentage of GCs at G0/G1 phase of the cell cycle. Additionally, overexpression of KISS1 could increase the mRNA levels of Star, CYP17, 3B-HSD, 17B-HSD of estrogen synthesis signaling pathway, significantly increase the concentration of E2 in the supernatant of the cultured GCs, and up-regulate the mRNA expression levels of ESR1 and ESR2. These results suggested that KISS1 might suppress cell apoptosis through activating the PI3K signaling pathway and stimulate synthesis of E2 via boosting the estrogen synthesis signaling pathway. This study would be of great interests for exploring the biological functionalities of KISS1 in the folliculogenesis and sex steroid production of the ovaries in mammals.