Recent studies suggest that microglia over‐expressing mutant human superoxide dismutase (mSOD1^G93A) may contribute to motoneuron death in a transgenic mouse model of familial amyotrophic lateral sclerosis. To further assess the relative neurotoxicity of wild‐type microglia, mSOD1^G93A microglia, and microglia over‐expressing wild‐type human SOD1, we used primary cultures of microglia and motoneurons in the presence and absence of lipopolysaccharide stimulation. Following activation with lipopolysaccharide, mSOD1^G93A microglia released more nitric oxide, more superoxide, and less insulin‐like growth factor‐1 than wild‐type microglia. In microglia/motoneuron co‐cultures, mSOD1^G93A microglia induced more motoneuron death and decreased neurite numbers and length compared with wild‐type microglia. Mutant SOD1^G93A microglia also induced more motoneuron injury than microglia over‐expressing wild‐type human SOD1 in microglia/motoneuron co‐cultures. Motoneuron survival was inversely correlated with nitrate + nitrite concentrations in mSOD1^G93A co‐cultures, suggesting the important role of nitric oxide in microglia‐induced motoneuron injury. Thus, relative to wild‐type microglia, mSOD1^G93A microglia were more neurotoxic and induced more motoneuron injury than similarly treated wild‐type microglia.