Abstract Background Amyotrophic lateral sclerosis (ALS) is a devastating late onset neurodegenerative disorder that is characterised by the progressive loss of upper and lower motor neurons. The mechanisms underlying ALS pathogenesis are unclear; however, there is emerging evidence the innate immune system, including components of the toll-like receptor (TLR) system, may drive disease progression. For example, toll-like receptor 4 (TLR4) antagonism in a spontaneous ‘wobbler mouse’ model of ALS increased motor function, associated with a decrease in microglial activation. This study therefore aimed to extend from these findings and determine the expression and function of TLR4 signalling in hSOD1 G93A mice, the most widely established preclinical model of ALS. Findings TLR4 and one of its major endogenous ligands, high-mobility group box 1 (HMGB1), were increased during disease progression in hSOD1 G93A mice, with TLR4 and HMGB1 expressed by activated microglia and astrocytes. hSOD1 G93A mice lacking TLR4 showed transient improvements in hind-limb grip strength and significantly extended survival when compared to TLR4-sufficient hSOD1 G93A mice. Conclusion These results suggest that enhanced glial TLR4 signalling during disease progression contributes to end-stage ALS pathology in hSOD1 G93A mice.