Gene expression in fungi by ambient pH is regulated via a conserved signalling cascade whose terminal component is the zinc finger transcription factor PacC/Rim1p. We have identified a pacC orthologue in the vascular wilt pathogen Fusarium oxysporum that binds the consensus 5'-GCCAAG-3' sequence and is proteolytically processed in a similar way to PacC from Aspergillus nidulans. pacC transcript levels were elevated in F. oxysporum grown in alkaline conditions and almost undetectable at extreme acidic growth conditions. PacC+/- loss-of-function mutants displayed an acidity-mimicking phenotype resulting in poor growth at alkaline pH, increased acid protease activity and higher transcript levels of acid-expressed polygalacturonase genes. Reintroduction of a functional pacC copy into a pacC+/- mutant restored the wild-type phenotype. Conversely, F. oxysporum merodiploids carrying a dominant activating pacCc allele had increased pacC transcript and protein levels and displayed an alkalinity-mimicking phenotype with reduced acid phosphatase and increased alkaline protease activities. PacC+/- mutants were more virulent than the wild-type strain in root infection assays with tomato plants, whereas pacCc strains were significantly reduced in virulence. We propose that F. oxysporum PacC acts as a negative regulator of virulence to plants, possibly by preventing transcription of acid-expressed genes important for infection.