KIF3A and KIF3B are two N-terminal motor proteins belonging to the kinesin-II superfamily that play essential roles in spermiogenesis. To understand the roles played by KIF3A/3B during spermatogenesis of large yellow croaker Larimichthys crocea, we studied the testis characteristics at different developmental stages of L. crocea, and determined the spatiotemporal expression patterns of kif3a and kif3b during spermiogenesis. Quantitative real-time PCR (qRT-PCR) showed that the overall trends of kif3a/3b mRNA abundance during testis development are similar. From stage II to stage V, kif3a/3b mRNA abundances first increased and then fell after reaching a peak at stage IV. Interestingly, the mRNA abundances of both genes at stage V were higher than those at stages II and III. In addition, it is worth of noting that kif3b mRNA abundance was higher than that of kif3a at all stages. Fluorescence in situ hybridization results revealed that kif3a/3b mRNA abundance dynamics were consistent with the migration of mitochondria, the deformation of nucleus, and the formation of tail. The mRNA hybridization signals of both genes first appeared either around the nuclear periphery or on the side of the nuclei, then appeared at one side of nuclei, and finally were mainly on the tail during spermiogenesis. Our findings contributed to better understanding the molecular mechanisms of spermiogenesis in fish; and suggested that KIF3A and KIF3B may participate in the intracellular transport of mitochondria, nuclear deformation, and the formation of tail during the spermiogenesis in L. crocea.