Multidrug resistance in Saccharomyces cerevisiae is frequently associated with gain-of-function mutations in zinc finger-containing transcription factors Pdr1p and Pdr3p. These regulatory proteins activate the expression of several ATP-binding cassette transporter genes, leading to elevated drug resistance. Here, we report that loss of the type 2A-related serine/threonine protein phosphatase Sit4p renders yeast cells sensitive to cycloheximide, azoles, daunorubicin and rhodamine 6G. This effect is a consequence of the decreased transcriptional levels of mainly PDR3 and its target genes, PDR5, SNQ2 and YOR1, which encode multidrug efflux pumps. The multidrug sensitivity of sit4 mutant cells is suppressed by the PDR1-3 mutant allele, which encodes a hyperactive form of Pdr1p. Sit4p is known to associate with regulatory proteins Sap155p, Sap4p, Sap185p and Sap190p. We found that the sap155 mutant strain is sensitive to azoles, but not to cycloheximide, while the sap155sap4 and sap185sap190 mutant strains are sensitive to both drugs. This finding indicates that the Sit4p-Sap protein complex subtly modulates the expression of drug efflux pumps. Drug resistance conferred by the expression of the Candida albicans CDR1 gene, an ortholog of PDR5 in S. cerevisiae, is also positively modulated by Sit4p. These data uncover a new regulatory pathway that connects multidrug resistance to Sit4p function.