Spermiogenesis is a highly ordered and complex process in the male germ cell differentiation. The microtubule-based motor proteins KIF3A and KIF3B are required for the progression of the stages of spermiogenesis. In this study, the main goal was to determine whether KIF3A and KIF3B have a key role in spermiogenesis in Palaemon carincauda. The complete cDNA of KIF3A/3B from the testis of P. carincauda was cloned by using PCR and rapid amplification of cDNA ends (RACE). The predicted secondary and tertiary structures of KIF3A/3B contained three domains which were the: a) head region, b) stalk region, and c) tail region. Real-time quantitative PCR (qPCR) results revealed that KIF3A and KIF3B mRNAs were obtained for all the tissues examined, with the greatest gene expression in the testis. In situ hybridization indicated the KIF3A and KIF3B mRNAs were distributed in the periphery of the nuclear in the early spermatid of spermiogenesis. In the middle and late spermatid stages, KIF3A and KIF3B mRNAs were gradually upregulated and assembled to one side where acrosome biogenesis begins. In the mature sperm, KIF3A and KIF3B mRNAs were distributed in the acrosome cap and spike. Immunofluorescence studies indicated that KIF3A, tubulin, mitochondria, and Golgi were co-localized in different stages during spermiogenesis in P. carincauda. The temporal and spatial gene expression dynamics of KIF3A/3B indicate that KIF3A and KIF3B proteins may be involved in acrosome formation and nucleus shaping. Moreover, these proteins can transport the mitochondria and Golgi that facilitate acrosome formation in P. carincauda.