Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca(superscript 2+) handling in PC3 cells. AC at concentrations of 5-50μg/ml did not affect cell viability, but at 100-200μg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200μg/ml did not alter basal [Ca(superscript 2+)](subscript i), but at a concentration of 25μg/ml decreased the [Ca(superscript 2+)](subscript i) increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25μg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200μg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca(superscript 2+)](subscript i), caused apoptosis via pathways unrelated to [Ca(superscript 2+)](subscript i) signal and phosphorylation of ERK, JNK and p38 MAPKs.