Rab GTPase is required for vesicle-vacuolar fusion during the vacuolar biogenesis in fungi. Rab GTPase-encoding gene, pcvA, was cloned from Penicillium chrysogenum: it contained five introns and its predicted protein contained the conserved Rab GTPase domain involved in GTP-binding and hydrolysis. Over-expression of pcvA significantly stimulated the vesicle-vacuolar fusion but repressed the conidiation and decreased conidial tolerance against thermal stress. Penicillin production was decreased in the pcvA over-expressed strain suggesting that pcvA is involved in vesicle-vacuolar fusion participates in the penicillin biosynthesis in P. chrysogenum.