Multifactorial and multistep alterations are involved in acquired fluconazole (FLC) resistance in Candida albicans. In this study, a FLC-resistant C. albicans strain was obtained by serial cultures of a FLC-susceptible C. albicans strain in incrementally increasing concentrations of FLC. The comparative proteomic study, confirmed by real-time RT-PCR, was performed with the susceptible parental strain and the resistant daughter strain to identify proteins altered during the development of FLC resistance. Our analysis of the differentially expressed proteins identified 22 different proteins, most of which were related to energy metabolisms (e.g., Pgk1, Fba1, and Adh1), and some of which have been previously identified as being involved in FLC resistance in C. albicans (e.g., Ald5, Cdc19, and Gap1). Functional analysis revealed lower intracellular ATP level and mitochondrial membrane potential, less endogenous reactive oxygen species generation in response to antifungal agents, and identical susceptibility to exogenous hydrogen peroxide, heat, and hyperosmotic shock in the resistant strain compared with the susceptible strain. Our results suggest that a metabolism shift might contribute to FLC resistance in C. albicans.