To clone a novel major facilitator superfamily (MFS, a large protein family with diverse physiological functions in all kingdoms) transporter gene, Pdmfs2, and characterize its function in Penicillium digitatum. A novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. The full-length DNA of Pdmfs2 had a 1590 bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7 mu g prochloraz/ml. The deletion of Pdmfs2 (Delta Pdmfs2) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value (the concentration of prochloraz producing 50 % growth inhibition) compared with the PdHS-F6 or complementation strain (COPdmfs2). The Delta Pdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent. Pdmfs2 is the second MFS transporter gene in P. digitatum and is required for prochloraz resistance, conidiation and full virulence.