It has been hypothesized that the dysregulation of transactive response DNA-binding protein-43 (TDP-43) in neurons is closely linked to the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. However, it remains undefined whether the dysregulation of TDP-43 in non-neuronal cells, such as glial cells, contributes to the pathogenesis of these neurodegenerative diseases. Primarily using HeLa cells, we show that a low-grade overexpression of TDP-43, 2- to 5-fold greater than endogenous expression, which is thought to mimic the gain of function of TDP-43, induced cell cycle arrest at the G2/M phase and cell death in cultured non-neuronal cells. Since the activation of p53 may induce G2/M arrest and/or cell death in many abnormal situations, we examined the mechanism underlying G2/M arrest from the standpoint of p53 regulation. It was determined that the TDP-43-induced G2/M arrest was attenuated, while TDP-43-induced death was not attenuated, in cells in which the p53 function was compromised. These data collectively indicate that TDP-43 causes G2/M arrest in a partially p53-dependent manner and it causes cell death in a p53-independent manner in cycling cells. Because it is likely that the impaired proliferation in glial cells causes a decrease in the neuron-supporting ability, these findings further suggests that the gain of function of TDP-43 may cause neurotoxicity by inducing cell cycle arrest and death in glial cells.