Spore germination is the first step for fungal pathogens to infect host plants. The pH value, as one of the most important environmental parameters, has critical influence on spore germination. In this study, effects of ambient pH on spore germination were determined by culturing spores of Penicillium expansum in medium with pH values at 2.0, 5.0 and 8.0, and involved mechanisms were further investigated through methods of comparative proteomics. The results demonstrated that spore germination of P. expansum was obviously inhibited at pH 2.0 and 8.0. Using quadrupole time-of-flight tandem mass spectrometer, 34 proteins with significant changes in abundance were identified. Among them, 17 proteins were related to protein synthesis and folding, and most of them were down-regulated at pH 2.0 and 8.0. Accordingly, lower content of total soluble proteins and higher ratio of aggregated proteins were observed in spores at pH 2.0 and 8.0. In addition, it was found that ambient pH could affect intracellular pH and ATP level of P. expansum spores. These findings indicated that ambient pH might affect spore germination of P. expansum by changing intracellular pH and regulating protein expression. Further, impairing synthesis and folding of proteins might be one of the main reasons.