4,4-Diisothiocyanatostilbene disulfonic acid (DIDS), an antagonist of anion channel including voltage-dependent anion channel (VDAC), acts as both neurotoxicant and neuroprotectant, resulting in the controversy. VDAC contributes to neuronal apoptosis and is a candidate target protein of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂). Caspase-3 is activated during neuronal apoptosis caused by 15d-PGJ₂. In the present study, we ascertained whether DIDS was neuroprotective or neurotoxic in the primary culture of rat cortical neurons. Neuronal cell viabilities were primarily evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H tetrazolium bromide (MTT) reduction assay. Plasma membrane integrity and apoptosis were detected by the staining of propidium iodide (PI) and Hoechst33342, respectively. Alternatively, apoptosis was also measured by caspase-3 assay kit. DIDS did not prevent neurons from undergoing the 15d-PGJ₂-induced apoptosis. In contrast, DIDS caused neuronal cell death in a concentration-dependent manner by itself, confirming its neurotoxicity. The sublethal application of DIDS did not decrease MTT-reducing activity, increase caspase-3 activity, condense chromatin, allow PI to enter neuron and degenerate neuronal morphology significantly. Interestingly, DIDS enhanced the 15d-PGJ₂-induced neuronal apoptosis markedly under the sublethal condition. To our knowledge, this is the first report of synergistic effects of DIDS on the neurotoxicity of 15d-PGJ₂.