Endochondral bone formation of the developing cranial base is a complex process. This mechanism requires precise orchestration of many cellular events and cartilage matrix metabolism, such as proliferation, becoming round in shape, termination of proliferation, hypertrophic size-increase, and finally programmed cell death. Active formation and degradation of cartilage matrix take place, in which microtubules are involved for intracellular events; bone apposition follows these events. However, the involvement of microtubules during these changes in the developing cranial base has not been identified yet. Thus, we investigated the involvement of microtubules in the regulation of endochondral bone formation during cranial base development. Using tubulin-binding drug nocodazole, we examined the effects of altering the structure and function of microtubules during in vivo organ culture of the mouse cranial base. Cultured specimens were analyzed with HE staining, immunohistochemistry, and cell counting in order to study the morphological and molecular changes that occurred in the tissues. Disruption of the microtubular array by nocodazole reduced cells expressing proliferation marker Ki67, osteogenic marker BSP, and BMP4 within the sphenooccipital synchondrosis region; chondrocyte hypertrophy was ceased in the hypertrophic zone; degeneration of cartilage matrix and bone matrix apposition was inhibited in the ossification center of the basooccipital cranial base. Our data demonstrated that disruption of microtubules by nocodazole have multiple inhibitory effects on the sequential changes that occur during endochondral bone formation, suggesting the importance of normal microtubule-polymerization in cranial base development.