Auranofin is a lipophilic gold compound with anti-inflammatory and immunosuppressive properties. This compound also exerts antiproliferative effects in several human cancer cell lines. Although auranofin induces apoptosis in human cancer cells, the underlying mechanisms remain unclear. This study investigated auranofin-mediated inhibition of cell growth and induction of mitochondrial apoptosis in PC3 human prostate cancer cells. Treatment with auranofin significantly inhibited cell viability with an IC50 value of 2.5 μM after 24 h. In particular, when cells were treated with 2.5 μM auranofin, there was a 2.2-fold increase in apoptotic cells compared to untreated cells. Auranofin activated caspase-3 and -8 in a concentration-dependent manner and decreased the levels of mitochondrial anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, auranofin enhanced oligomerization of the voltage-dependent anion channel (VDAC) in a concentration- and time-dependent manner. Interestingly, auranofin significantly enhanced annexin A5 mRNA and protein expression and promoted annexin A5 translocation into the mitochondria. In order to characterize the function of annexin A5 in auranofin-induced mitochondrial apoptosis, annexin A5 was depleted using siRNA. Annexin A5 siRNA suppressed auranofin-mediated annexin A5 expression and VDAC oligomerization. Accordingly, annexin A5 depletion rescued auranofin-induced apoptosis, which may be mediated by caspase-3 activation. In conclusion, the present findings suggest that auranofin induces mitochondrial apoptosis through induction of annexin A5 expression and translocation as well as VDAC oligomerization in human prostate cancer cells.