Aberrant or excessive pro-inflammatory microglial activation contributes to many neurodegenerative diseases. This activation is modeled in vitro by combined exposure to the Toll-like receptor 4 (TLR4)-activating molecule lipopolysaccharide (LPS) and the cytokine interferon-gamma (IFN-γ), which cause microglia to adopt a neurotoxic state. Inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production, mitochondrial electron transport chain (ETC) dysfunction, and NLRP3 inflammasome-dependent caspase-1 activation are implicated in the pro-inflammatory activation. The mitochondrial ETC dysfunction, which includes large decreases in multiple ETC complex subunits, is thought to depend on NO production. However, the precise mechanisms of subunit loss are unclear. Here, we tested the hypothesis that both TLR4-dependent caspase-1 protease activation and NO production are required for mitochondrial ETC subunits loss in proinflammatory microglia. Using an OXPHOS antibody cocktail, we examined the level of ETC complex proteins by western blot at 18 hours post LPS+IFN-γ stimulation in wild type (WT) and Nos2 knockout HAPI mouse microglial cells ± caspase-1 inhibitor and/or TLR4 inhibitor. Complex I subunit NDUFB8, Complex II subunit SDHB, and Complex IV subunit COX1 were all reduced relative to α-tubulin or total protein in LPS+IFN-γ-activated WT HAPI cells whereas Complex III UQCRC2 subunit and Complex V ATP5A subunit were unchanged. CRISPR knockout of Nos2 and inhibition of NO production in WT cells each rescued the decrease in Complex II and IV subunits but did not preserve the Complex I subunit NDUFB8. Addition of caspase-1 inhibitor VX765 or TLR4 antagonist TAK-242 failed to rescue any of the ETC complex proteins. However, each inhibitor moderately reduced iNOS protein expression and their combined addition led to a partial rescue of COX1. Unexpectedly, iNOS expression was partially suppressed by caspase-1 inhibitor and, also, a decreased level of the caspase-1 p20 active fragment was detected when NO production was prevented by the iNOS inhibitor 1400W or by Nos2 ablation. These results suggest that there is a positive feedback loop between caspase-1 activation and nitric oxide production. In addition, findings indicate that mitochondrial ETC dysfunction is mediated by multiple mechanisms, as SDHB and COX1 but not Complex I subunit NDUFB8 were rescued by nitric oxide elimination and only COX1 loss was sensitive to the caspase-1-TLR4 inhibitor combination. Additional work is needed to elucidate the TLR4-, caspase-1-, and nitric oxide-independent mechanisms recruited by LPS+ IFN-γ that impair the mitochondrial ETC in microglia.