The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1Delta mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1-overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans-microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.