Sphingolipids have been reported to regulate the growth and death of mammalian and yeast cells, but their precise mechanisms are unknown. In this paper, it was shown that the deletion of the oxysterol binding protein homologue 3 (OSH3) gene confers hyper resistance against ISP-1, an inhibitor of sphingolipid biosynthesis, in the yeast Saccharomyces cerevisiae. Furthermore, the overexpression of the ROK1 gene, which directly binds to Osh3p, conferred resistance against ISP-1, and the deletion of the KEM1 gene, which regulates microtubule functions, exhibited ISP-1 hypersensitivity. And yet, an ISP-1 treatment caused an abnormal mitotic spindle formation, and the ISP-1-induced cell cycle arrest was rescued by the deletion of the OSH3 gene. Taken together, it is suggested that the expression levels of the OSH3 gene influence the ISP-1 sensitivity of S. cerevisiae, and the sphingolipids are necessary for normal mitotic spindle formation in which the Osh3p may play a pivotal role.