Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease most commonly treated with riluzole, a small molecule considered to act at least in part via modulation of glutamatergic neurotransmission. However, riluzole affords only a modest extension of lifespan for people living with ALS and its precise mechanisms of action remain largely unclear. Likewise, the vast majority of ALS cases are characterised by the pathological accumulation of cytoplasmic TDP-43, but the effects of riluzole in an in vivo model of ALS with disease-reminiscent TDP-43 pathology have not been thoroughly studied. We therefore tested the effects of daily riluzole treatment on TDP-43 pathology and disease onset and progression in transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons of the brain and spinal cord (NEFH-tTA/tetO-hTDP-43ΔNLS, ‘rNLS’, mice). We found that treatment of rNLS mice with riluzole beginning from the first day of hTDP-43ΔNLS expression failed to alter disease onset, disease-associated weight loss or performance on multiple motor behavioural tasks over a 6-week period. Riluzole treatment also did not alter soluble or insoluble TDP-43 protein levels or TDP-43 phosphorylation in rNLS mice. By quantifying levels of key proteins involved in glutamatergic signalling, we identified a dramatic loss in GluA3 protein in the rNLS mice after disease onset, however riluzole was unable to ameliorate this disease-associated molecular phenotype. Finally, we assessed the ability of riluzole to affect disease in a long-term post-disease onset study in rNLS mice, and found that riluzole similarly had no effect on progression of late-stage disease or animal survival. Together, our findings demonstrate that the rNLS mouse model recapitulates glutamatergic receptor alterations reminiscent of ALS, but the approved ALS therapeutic riluzole has no effect on disease phenotypes in these animals. These studies suggest that strategies directly targeting disease-relevant pathways, such as accumulation of TDP-43 pathology, may be needed for development of more effective ALS treatments.