ABSTRACT Nettle showed several benefits for animals as an unconventional feed source, but it is difficult to ensile in order to be better utilized. Recently, nettle has been utilized to alternate alfalfa as ruminant feed, but it is usually poor in ensiling quality. To determine the endogenous component influencing ensiling, we investigated the effect of Pediococcus on the characteristics of nettle silage and the mechanism of action of these substances against Pediococcus. Inoculation with Pediococcus pentosaceus decreased the relative abundance of Clostridium sensu stricto 15 by 85.95% in the middle fermentation stage of nettle silage (30 d), with a 24.74% decrease in the ammonia content (P＜0.05). In the correlation analysis, 4-pentenoic acid showed an extremely significant negative correlation with Pediococcus spp. in nettle silage (P < 0.001). After 4-pentenoic acid treatment, the most downregulated proteins were involved in the ribosome pathway (30 differentially expressed proteins), and then in the glycolysis/gluconeogenesis, pyruvate metabolism, and fatty acid synthesis pathways (particularly for accA, accD, and fabG). The mechanism of action of 4-pentenoic acid against P. pentosaceus mainly involves inhibition of fatty acid synthesis and decreased the expression of acid tolerance proteins. The present study will give new insights into silage fermentation and provide new clues for better ensiling of nettle.IMPORTANCENettle has attracted the attention of scientists due to its several benefits for animals as non-conventional feed sources. However, as for challenge, nettle is difficult to ensile (poor quality), which is an obstacle for nettle use. In the present manuscript, we investigated the effect of Pediococcus on the characteristics of nettle silage and clarified the mechanisms of 4-pentenoic acid against Pediococcus. Our findings suggested that P. pentosaceus could improve nettle silage quality at a significant level through decreased production of ammonia (decline percentage was 21.41%–31.73%) during ensiling, while it could not well improve the quality of nettle silage due to the interference effect of 4-pentenoic acid as an antibacterial substance. The mechanism of 4-pentenoic acid against P. pentosaceus was mainly through inhibition of fatty acid synthesis (fabG) and expression of acid tolerance protein (accA), resulting in destruction of the cell wall in P. pentosaceus. Our finding could give a new clue for better use of nettle silage.