In order to explore the dynamic mechanisms during spermatogenesis of the penaeid prawns, the full length of kifc1 was cloned from testis cDNA of Penaeus japonicus through RACE. Both semi-quantitative RT-PCR and Western blot results indicated that KIFC1 was extensive expressed in different tissue of P. japonicus. Compared with other tissue, the highest expression of KIFC1 occurred in the testis. According to the immunofluorescence results, the KIFC1 protein was detected at each stage of whole process of spermatogenesis. In the spermatogonial phase, KIFC1 mainly dispersed in cytoplasm and co-localized with microtubules, while abundant KIFC1 signal was detected in the nucleus of spermatocytes. At the early stage of spermatids, KIFC1 was transported from the nucleus into the cytoplasm, and it assisted microtubule assembly onto one side of the nucleus. Finally, in mature sperm, it was weakly expressed in the acrosome. This implies that KIFC1 may participate in the mitosis of spermatogonia, meiosis of spermatocyte, and acrosome formation during spermiogenesis; it may also play functions in acrosome maintaining in mature sperm. In addition, the results of KIFC1 knockdown by dsRNA injection in vivo reveal that decreased KIFC1 expression may induce aberrant microtubule assembly, and it leads to spermatogonia and spermatocyte apoptosis.