pH is a key player in the control of fungal pathogenicity. We previously found that extracellular pH governs pathogenicity in the plant pathogen Fusarium oxysporum by reprogramming phosphorylation levels of mitogen-activated protein kinases (MAPKs). The molecular events underlying the pH response are currently unknown. Here we identify intracellular pH (pHi) as a key signal regulating MAPK activity in F. oxysporum. Using the ratiometric GFP-based pH sensor pHluorin, we found that F. oxysporum responds to extracellular alkalinisation and acidification with a transitory shift in pHi. Exogenous application of diethylstilbestrol (DES), a specific inhibitor of the plasma membrane H+-ATPase Pma1, induced a rapid and sustained decrease of pHi accompanied by rapid and transitory changes in MAPK phosphorylation, supporting the idea that pHi acts as a key switch controlling MAPK activity. To search for fungal proteins involved in pHi-mediated MAPK regulation, we screened a subset of acid-sensitive mutants from the yeast deletion library for loss of DES-triggered MAPK phosphorylation. This identified a number of candidates functioning in conserved cellular processes such as lipid metabolism, endocytosis or V-ATPase function, many of which have predicted orthologs in Fusarium. Understanding how pHi regulates MAPK signaling may reveal new ways to control fungal growth, development and pathogenicity.