To explore fluconazole’s effect mechanism, we investigated the changes of viability rate, reactive oxygen species (ROS), mitochondrial membrane potential (△Ψm) and cell cycle of Candida tropicalis after treatment with fluconazole. The minimum inhibitory concentration (MIC) of the clinical isolates Candida tropicalis to fluconazole were tested by NCCLS M27-A microdilution method. After treatment wth different concentration of fluconazole, viability rate, the intracellular accumulation of ROS, the loss of mitochondrial membrane potential △Ψm and cell cycle of Candida tropicalis were detected with flow cytometry, respectively After treatment with fluconazole, there were no significant variation among viability rate, ROS, mitochondrial membrane potential △Ψm and cell cycle in flu-conazole-resistant strains, but a decrease of mitochondrial membrane potential △Ψm and viability rate, an increase of ROS accumulation were detected in a time-dose-dependent manner in flucona-zole-susceptibile strains. A majority of Candida tropicalis were arrested in G2/M phase and apoptosis peak was seen. Free radicals scavenger glutathione inhibited ROS production, prevented G2/M arrest and decreased apoptosis in fluconazole-susceptibile strains. According to it, fluconazole maybe induce intracellular accumulation of ROS and decrease of mitochondrial membrane potential △Ψm, which could result in apoptosis of Candida tropicalis.