Post-insemination molecular interactions involving sperm are an ideal system for linking genotype, phenotype, and fitness. Nematode sperm contain subcellular vesicles known as membranous organelles (MOs) that fuse with the cell membrane upon sperm activation to release their soluble contents into the extracellular space. The second most abundant protein group in the MOs is the conserved Nematode-Specific Peptide family, group F (NSPF) gene family. We hypothesize that these proteins contribute to seminal fluid upon MO fusion and are part of post-insemination reproductive tract dynamics. We used experimental evolution to compete the wildtype allele against a deletion allele in 10 replicate populations. We calculated a significant mean selective disadvantage of 0.1% for the deletion allele, which indicated that the NSPF genes are an evolutionarily important component of male reproductive success. This conclusion was reinforced by qualitative trends from lower powered single-generation male fertility assays. We then characterized the functional location of NSPF proteins during fertilization using whole- worm immunostaining of a His-tagged nspf-1 transgene. Throughout male development, we found that NSPF presence and abundance was correlated with reproductive maturity in males, localizing to the seminal vesicle during L4 and to the spicules throughout adulthood. We confirmed that NSPF proteins are transferred to females during mating and hypothesize that proteins adhere to the spicules during transfer. NSPF proteins localize to the vulva and uterus lumen when transferred to mated females and in unmated adult hermaphrodites. These results suggest that the uterine localization of the NSPF proteins is likely a functional property of both male-derived sperm and self-sperm and not simply incidental to the point of transfer during mating. Our study demonstrates that nematodes use a novel mechanism for generating seminal fluid proteins and shows that the highly abundant NSPF proteins likely have a female-beneficial fitness effect.