Cell division is controlled by a multitude of protein enzymes, but little is known about roles of metabolites in this mechanism. Here, we show that pregnenolone (P5), a steroid that is produced from cholesterol by the steroidogenic enzyme Cyp11a1, has an essential role in centriole cohesion during mitosis. During prometa-metaphase, P5 is accumulated around the spindle poles. Depletion of P5 induces multipolar spindles that result from premature centriole disengagement, which are rescued by ectopic introduction of P5, but not its downstream metabolites, into the cells. Premature centriole disengagement, induced by loss of P5, is not a result of precocious activation of separase, a key factor for the centriole disengagement in anaphase. Rather, P5 directly binds to the N-terminal coiled-coil domain of short-form of shugoshin 1 (sSgo1), a protector for centriole cohesion and recruits it to spindle poles in mitosis. Our results thus reveal a steroid-mediated centriole protection mechanism.