Throughout spermatogenesis, cellular cargoes including haploid spermatids required to be transported across the seminiferous epithelium, either towards the microtubule (MT) plus (+) end near the basement membrane at stage V, or to the MT minus (-) end near the tubule lumen at stages VI-VIII, of the epithelial cycle. Furthermore, preleptotene spermatocytes differentiated from type B spermatogonia are transported across the Sertoli cell blood-testis barrier (BTB) to enter the adluminal compartment. Few studies, however, were conducted to explore the function of MT-dependent motor proteins to support spermatid transport during spermiogenesis. We thus examined the role of MT-dependent and microtubule plus (+) end directed motor protein KIF15 in the testis. KIF15 displayed a stage-specific expression across the seminiferous epithelium, associated with MTs and appeared as aggregates on the MT tracks that aligned perpendicular to the basement membrane and laid across the entire epithelium. KIF15 also tightly associated with apical ES (ectoplasmic specialization), displaying strict stage-specific distribution, apparently being used to support spermatid transport across the epithelium. We used a loss-of-function approach by RNAi to examine the role of KIF15 in Sertoli cell epithelium in vitro to examine its role in cytoskeletal-dependent Sertoli cell function. It was noted that KIF15 knockdown (KD) by RNAi that reduced KIF15 expression by ~70% in Sertoli cells with an established functional tight junction (TJ)-barrier impeded the barrier function. This effect was mediated through remarkable changes in the cytoskeletal organization of MTs, but also actin-, vimentin-, and septin-based cytoskeletons, illustrating KIF15 exerts its regulatory effects well beyond microtubules.