Effects of rhizosphere properties on the rhizosphere priming effect (RPE) are unknown. This study aimed to link species variation in RPE with plant traits and rhizosphere properties. Four C3 species (chickpea, Cicer arietinum; field pea, Pisum sativum; wheat, Triticum aestivum; and white lupin, Lupinus albus) differing in soil acidification and root exudation, were grown in a C4 soil. The CO2 released from soil was trapped using a newly developed NaOH-trapping system. White lupin and wheat showed greater positive RPEs, in contrast to the negative RPE produced by chickpea. The greatest RPE of white lupin was in line with its capacity to release root exudates, whereas the negative RPE of chickpea was attributed to its great ability to acidify rhizosphere soil. The enhanced RPE of field pea at maturity might result from high nitrogen deposition and release of structural root carbon components following root senescence. Root biomass and length played a minor role in the species variation in RPE. Rhizosphere acidification was shown to be an important factor affecting the magnitude and direction of RPE. Future studies on RPE modelling and mechanistic understanding of the processes that regulate RPE should consider the effect of rhizosphere pH.