DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF ± DIDS (40 or 400 µM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N-terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.