The manifestation of virulence traits in Cryptococcus neoformans is thought to rely on intracellular transport, a process not fully explored in this pathogenic fungus. Through interaction cloning, we identified a multi-modular protein, Cin1 (cryptococcal intersectin 1), whose domain structure is similar to that of the human endocytic protein ITSN1. Cin1 contains an N-terminal EH domain, a central coiled-coil region, a WH2 domain, two SH3 domains and a C-terminal RhoGEF (DH)-PH domain. Interestingly, alternative mRNA splicing resulted in two Cin1 isoforms, and Cin1 homologues are also restricted to basidiomycetous fungi. Disruption of the CIN1 gene had a pleiotropic effect on growth, normal cytokinesis, intracellular transports and the production of several virulence factors. Additionally, Cin1 interacts with cryptococcal Cdc42 and Wsp1 (a WASP homologue) proteins in vitro, suggesting a conserved role in the regulation of the actin cytoskeleton. However, deletion of RhoGEF or SH3 and RhoGEF domains did not result in any phenotypic changes, suggesting that functional redundancy exists in proteins containing similar domains or that the activities by other domains are necessary for Cin1 function. Our study presents the first evidence of a multi-modular protein whose function in intracellular transport underlies the growth, differentiation and virulence of a pathogenic microorganism.