Although alpha-L-fucosidase has been shown to be important during reproduction in many diverse organisms, the protein’s specific role remains unclear. To our knowledge, this would be the first report of alpha-L-fucosidase in zebrafish milt. Moreover, the utilization of zebrafish will allow for an evaluation of alpha-L-fucosidase’s potential role in embryonic development. Alpha-L-fucosidase activity in zebrafish sperm was quantified using the substrate 4-methylumbelliferyl-alpha-L-fucopyranoside (4-MU-fuc) to produce a fluorometric product known as 4-methyl-umbelliferone (4-MU). Fluorometric results suggested the presence of alpha-L-fucosidase in zebrafish milt. Partitioning assays were conducted to evaluate the presence of alpha-L-fucosidase in the sperm cells versus the liquid component of milt. Although not significant, a higher amount of alpha-L-fucosidase was detected in the pellet compared to the supernatant fraction. Treatment of zebrafish samples with deoxyfuconojirimycin (DFJ), a potent and specific inhibitor of alpha-L-fucosidase, inhibited enzyme activity. Egg extracts were prepared and evaluated for the presence of alpha-L-fucosidase and revealed minimal detection. In vitro fertilization experiments were conducted to evaluate embryonic success over 72 hours in the absence and presence of DFJ. A decrease in fertilization success was noted when comparing untreated versus DFJ treated experiments 0.75-2 hours post insemination (hpi). In both untreated and DFJ treated experiments, embryos successfully developed through 72 hpi. Understanding the role of alpha-L-fucosidase in reproduction could provide an understanding that can be translated to improving infertility in humans.