Phelligridin D (Phe D) is a compound isolated from Phellinus baumii, which is known for various biological activities. In this study, the authors examined the effect of Phe D on boar spermatozoa for its potential application in assisted reproductive technology for mammals. Sperm motility and deubiquitinylating activity significantly decreased when boar spermatozoa were incubated with Phe D (>0.5 µM). The fluorescence intensities of dead sperm, and reactive oxygen species production increased after sperm incubation in the presence of Phe D. Although Phe D is associated with antioxidant and free radical scavenging activity, sperm viability deteriorated after its addition. This could lead to fertilization failure, including that following artificial insemination or in vitro fertilization. Phe D might have other biological functions in spermatozoa, and therefore requires additional studies in the future.