The phenolic compound 2,5-dimethylphenol is a natural product. 2,5-Dimethylphenol has been shown to affect rat hepatic and pulmonary microsomal metabolism. However, the effect of 2,5-dimethylphenol on Ca2+ signaling and cyotoxicity has never been explored in any culture cells. This study explored the effect of 2,5-dimethylphenol on cytosolic free Ca2+ levels ([Ca2+]i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500 μM and 1000 μM evoked [Ca2+]i rises in a concentration-dependent manner. This Ca2+ signal was inhibited by approximately half by the removal of extracellular Ca2+. 2,5-Dimethylphenol-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Pretreatment with the protein kinase C (PKC) inhibitor GF109203X, nifedipine or the store-operated Ca2+ entry inhibitors (econazole or SKF96365) inhibited 2,5-dimethylphenol-induced Ca2+ signal in Ca2+-containing medium by ∼30%. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin in Ca2+-free medium abolished 2,5-dimethylphenol-induced [Ca2+]i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca2+]i rises by ∼80%. 2,5-Dimethylphenol killed cells at concentrations of 350–1000 μM in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid/AM (BAPTA/AM) did not prevent 2,5-dimethylphenol’s cytotoxicity. Together, in PC3 cells, 2,5-dimethylphenol induced [Ca2+]i rises that involved Ca2+ entry through PKC-regulated store-operated Ca2+ channels and PLC-dependent Ca2+ release from the endoplasmic reticulum. 2,5-Dimethylphenol induced cytotoxicity in a Ca2+-independent manner.