Autophagy is a conserved pathway essential for granulosa cell homeostasis and follicular development. Spermidine, the sole substrate for eIF5A hypusination (eIF5Ahyp), promotes autophagy and exerts diverse protective effects, including antioxidant and anti-aging activities. This study demonstrates that the spermidine-eIF5Ahyp axis influences autophagy in porcine ovarian granulosa cells by regulating the expression of the downstream protein TUFM. These findings provide mechanistic insights into autophagic regulation in reproductive cells and highlight potential targets for enhancing mammalian fertility.