Microglial cells have been attributed both neuroprotective and neurodegenerative roles in cerebral ischemia. This study presents an immunotoxic method for depletion of microglia from mouse hippocampal slice cultures and the effect of this on ischemia-like oxygen–glucose deprivation (OGD). For microglial depletion hippocampal slice cultures were exposed for 7 days to saporin coupled to an antibody against the microglial receptor Mac1 (Mac1–sap). When subjected to OGD immediately thereafter, resulting neurodegeneration was quantified as cellular uptake of propidium iodide (PI). Cultures were processed 1, 7 or 14 days after OGD for general cell staining and immunohistochemistry for neuronal, astroglial and microglial markers. Following Mac1–sap treatment there was a near total loss of microglia, these microglia-depleted cultures displayed a significant increase in PI uptake and astrogliosis 1 day after OGD compared to non-depleted cultures. In cultures surviving 7 and 14 days after OGD there was a decrease in PI uptake compared to 1 day after OGD. At 7 and 14 days after OGD the differences in Mac1–sap treated or non-treated cultures were still noticeable in terms of more neuron loss in cultures deprived of microglia, while the astroglial reactivity seemed to equalize. Based on the finding that depletion of microglia significantly increased OGD-induced CA1 pyramidal cell degeneration, we conclude that microglia at least in the initial phase of injury exert a neuroprotective role in mouse hippocampal slice cultures.