Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death in vitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death in vitro and in vivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role. •NF-κB is activated in ALS, predominantly in microglia•Inhibition of microglial NF-κB delays disease progression by 47% in SOD1-G93A mice•Inhibition of NF-κB dampens proinflammatory microglial activation•Constitutive activation of microglial NF-κB induces pathological hallmarks of ALS Frakes et al. demonstrate NF-κB is activated in microglia to kill motor neurons in ALS. These findings identify a novel therapeutic target to slow the progression of ALS and possibly other neurodegenerative disease in which microglial activation plays a role.