The brominated flame retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), is well documented to exert potential negative impacts on different marine biota. However, the responsible mechanism remains unknown. The rainbow trout gonadal cell line RTG-2 was used as a model, and the mechanism and pathway underlying BDE-47 (6, 12.5 and 25 μM)-induced apoptosis and toxicity were examined in vitro. Apoptosis occurred in the RTG-2 cells exposed to BDE-47 in a clear concentration-dependent manner. The morphology of the mitochondrial alterations was observed using transmission electron microscopy. BDE-47 exposure decreased the cellular mitochondrial membrane potential, increased the cytochrome c released into the cytoplasm and elevated Fas protein expression. The mRNA expressions of Fas-associated death domain-containing protein (FADD), CHOP and GRP78 were also elevated, and similar increases were found in the activities of intracellular caspase-8, caspase-12, caspase-9 and caspase-3. These results indicated that the mitochondrial, endoplasmic reticulum and death-receptor pathways were involved in apoptosis in RTG-2 cells following BDE-47 exposure. ROS and Ca were responsible for these changes because their overproduction was detected prior to apoptosis. However, the addition of the ROS scavenger N-acetyl-l-cysteine (NAC) and the intracellular calcium chelator (acetoxymethyl)-1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) did not significantly alleviate the apoptosis rate. The results of the present study show that BDE-47 exposure induced apoptosis in RTG-2 cells via ROS- and Ca-mediated mitochondrial, endoplasmic reticulum and death-receptor apoptotic pathways.