Background Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Results To explore the pathogenic properties of mutant forms of TDP-43, we generated and characterized two mouse lines expressing human TDP-43 carrying the M337V mutation (hTDP-43M337V). We found hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, intranuclear and cytoplasmic phosphorylated TDP43 aggregates were frequently detected, and the levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased. Overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice. Conclusion While overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity.