The most common inherited form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motoneurons, is caused by dominant mutations in the ubiquitously expressed Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). Recent studies suggest that glia may contribute to motoneuron injury in animal models of familial ALS. To determine whether the expression of mutant SOD1 (mSOD1^G93A) in CNS microglia contributes to motoneuron injury, PU.1^−/− mice that are unable to develop myeloid and lymphoid cells received bone marrow transplants resulting in donor-derived microglia. Donor-derived microglia from mice overexpressing mSOD1^G93A, an animal model of familial ALS, transplanted into PU.1^−/− mice could not induce weakness, motoneuron injury, or an ALS-like disease. To determine whether expression of mSOD1^G93A in motoneurons and astroglia, as well as microglia, was required to produce motoneuron disease, PU.1^−/− mice were bred with mSOD1^G93A mice. In mSOD1^G93A/PU.1^−/− mice, wild-type donor-derived microglia slowed motoneuron loss and prolonged disease duration and survival when compared with mice receiving mSOD1^G93A expressing cells or mSOD1^G93A mice. In vitro studies confirmed that wild-type microglia were less neurotoxic than similarly cultured mSOD1^G93A microglia. Compared with wild-type microglia, mSOD1^G93A microglia produced and released more superoxide and nitrite+nitrate, and induced more neuronal death. These data demonstrate that the expression of mSOD1^G93A results in activated and neurotoxic microglia, and suggests that the lack of mSOD1^G93A expression in microglia may contribute to motoneuron protection. This study confirms the importance of microglia as a double-edged sword, and focuses on the importance of targeting microglia to minimize cytotoxicity and maximize neuroprotection in neurodegenerative diseases.