Cryptococcus neoformans is a significant human fungal pathogen, and its kinases, Hsl101 and Urk1, play pivotal roles in virulence during lung and brain infections. This study systematically investigates the effects of Hsl101 and Urk1 deletions on the proteomic and metabolomic profiles of C. neoformans. By generating deletion mutants for Hsl101, we identified profound alterations in the expression levels of 366 proteins and 421 metabolites, including those involved in oxidative phosphorylation and ATP synthesis. Similarly, deletion of Urk1 resulted in significant changes in the expression of 236 proteins and 366 metabolites, highlighting pathways such as steroid biosynthesis and starch and sucrose metabolism. These findings not only elucidate the regulatory roles of Hsl101 and Urk1 in the metabolic pathways of C. neoformans but also pave the way for potential therapeutic interventions targeting these kinases.