Systemic candidiasis is most commonly studied in animal models, particularly the murine intravenous (IV) challenge model, where infection with a virulent Candida albicans strain leads to increasing fungal kidney burdens and increasing pro-inflammatory cytokines in the kidneys. Based upon the finding that early renal levels of the chemokine KC correlate with infection outcome, a new in vitro model, utilising the murine renal M-1 cortical collecting duct epithelial cell line, was developed to evaluate virulence of C. albicans isolates and mutants, in attempts to reduce the number of mice used in C. albicans virulence studies, addressing the 3Rs. The epithelial cells were shown to respond only to live fungal cells, unlike immune cells, responding more robustly to hyphae rather than to cells growing as yeasts. We also demonstrate that non-albicans Candida species, which are attenuated in the mouse IV challenge model, are unable to elicit chemokine responses from mouse kidney epithelial cells, despite increasing the inoculums used. Renal epithelial cell responses observed in the new model reflect early events in the mouse model, with chemokines KC and MIP-2 produced in response to virulent C. albicans strains or mutants. This chemokine production correlates with C. albicans damage to epithelial cells. Some involvement of TLR4 signalling was demonstrated as blocking of TLR4 signalling reduced epithelial KC production, and it was demonstrated that the renal epithelial cells respond strongly to more complex glycan molecules. Using this new in vitro model we have confirmed that renal epithelial cells are able to discriminate between virulent and attenuated strains of C. albicans, allowing this model to be used as an initial screen for altered virulence and for investigating how renal epithelial cells detect the presence of pathogenic fungi.