Recent studies suggest that the inducible isoform of cyclooxygenase, COX‐2, promotes motor neuron loss in rodent models of ALS. We investigated the effects of PGE₂, a principal downstream prostaglandin product of COX‐2 activity, on motor neuron survival in an organotypic culture model of ALS. We find that PGE₂ paradoxically protects motor neurons at physiological concentrations in this model. PGE₂ exerts its downstream effects by signaling through a class of four distinct G‐protein–coupled E‐prostanoid receptors (EP1–EP4) that have divergent effects on cAMP. EP2 and EP3 are dominantly expressed in ventral spinal cord in neurons and astrocytes, and activation of these receptor subtypes individually or in combination also rescued motor neurons. The EP2 receptor is positively coupled to cAMP, and its neuroprotection was mimicked by application of forskolin and blocked by inhibition of PKA, suggesting that its protective effect is mediated by downstream effects of cAMP. Conversely, the EP3 receptor is negatively coupled to cAMP, and its neuroprotective effect was blocked by pertussis toxin, suggesting that its protective effect is dependent on Gi‐coupled heterotrimeric signaling. Taken together, these data demonstrate an unexpected neuroprotective effect mediated by PGE₂, in which activation of its EP2 and EP3 receptors protected motor neurons from chronic glutamate toxicity. Ann Neurol 2004;56:240–248