Technical Abstract: All-trans-retinoic acid (ATRA) is a vitamin A derivative that is important in neuronal patterning, survival, and neurite outgrowth. We investigated the relatively acute effects of ATRA (100 nM and 1 µM) on cell swelling in ischemic injury and on key features hypothesized to contribute to cell swelling including increased reactive oxygen species/reactive nitrogen species (ROS/RNS), depolarization of the inner mitochondrial membrane potential (''m), and increased intracellular calcium ([Ca2+]i). C6 glial cell cultures were subjected to 5 hr oxygen-glucose deprivation (OGD) and cell volume was determined at 90 min after the end of OGD. OGD increased cell volume by 43% but this increase was significantly attenuated by both doses of ATRA. OGD induced an increase in ROS/RNS production in the whole cell and mitochondria, as assessed by the fluorescent dyes CM-H2DCFDA and MitoTracker CM-H2-XROS at 30 min after the end of OGD. The increase in mitochondrial ROS, but not cellular ROS, was significantly attenuated by ATRA. OGD also induced a 67% decline in mitochondrial ''m but this decline was significantly attenuated by ATRA. OGD-induced increase in [Ca2+]i was also significantly attenuated by ATRA. Taken together with our previous results where calcium channel blockers reduced cell swelling, the effects of ATRA in attenuating swelling are possibly mediated through its effects in regulating [Ca2+]i. Considering the paucity of agents in attenuating brain edema in ischemia, ATRA has the potential to reduce brain edema and associated neural damage in ischemic injury.