Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons, which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS, and TDP-43 modulates several mitochondrial transcripts. In this study, we used induced pluripotent stem cell-derived motor neurons (iPS-MNs) from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient iPS-MNs, linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient iPS-MNs was detected, which correlated with downregulation of the motor protein complex, dynactin-1 (DCTN1)/dynein. Overexpression of DCTN1 in patient iPS-MNs significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS iPS-derived MNs with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport, which can be partially rescued by DCTN1 overexpression.