Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100 mu M silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K+ and accumulation of cytoplasmic and mitochondrial Ca2+. Among these phenomena, mitochondrial Ca2+ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca2+ in apoptosis by employing 20 mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca2+ signaling in C. albicans. (C) 2016 Elsevier Ltd. All rights reserved.