Phosphatidylinositol (PtdIns) transfer proteins (PITPs) mediate the exchange of PtdIns and phosphatidylcholine between membranes, a step required for a wide array of cellular processes including cell division. SMI-481 is an inhibitor of the yeast PITP Sec14, blocking both Sec14-mediated transfer of [3H]-PtdIns in vitro and growth (IC50s = 0.21 and 2.87 μM, respectively). Suppression of Sec14 expression leads to proportional reduction in SMI-481 inhibition, suggesting that SMI-481 acts specifically through Sec14. Moreover, SMI-481 does not affect [3H]-PtdIns transfer mediated by other yeast PITPs.