In the human fungal pathogen Cryptococcus neoformans, activation of the phosphate signalling and acquisition (PHO) pathway and production of the inositol pyrophosphate 5-PP-IP5 are essential for fungal dissemination to the host brain and the ensuing fatal meningitis. Whether the PHO pathway and 5-PP-IP5 are connected is unknown. We now demonstrate that 5-PP-IP5 and the PHO pathway intersect at the cyclin-dependent kinase (CDK) inhibitor (CKI) Pho81, to promote activation of the PHO pathway in C. neoformans. This is in contrast to the model yeast, Saccharomyces cerevisiae, where 5-PP-IP5 suppresses PHO pathway activation. Using site-directed mutagenesis of Pho81, Pho81 affinity capture using a custom-made 5-PP-IP5-conjugated resin, Pho81-CDK co-immunoprecipitation and LC-MS, we demonstrate that irrespective of phosphate status, cryptococcal 5-PP-IP5 stabilizes the CKI-CDK complex in vivo by binding to a lysine surface cluster within the SPX domain of CKI Pho81. Disrupting 5-PP-IP5-Pho81 interaction in vivo impairs Pho81-CDK association, leading to defective PHO pathway activation and complete loss of virulence in a mouse infection model. In summary, our study investigating the impact of disrupting a single 5-PP-IP5-SPX domain interaction on fungal virulence has identified a novel role for 5-PP-IP5 in stabilizing the CKI-CDK complex to promote, rather than suppress, PHO pathway activation and highlights evolutionary changes in PHO pathway regulation that promote deadly systemic infection by a fungal pathogen of medical significance.