Developmental neurotoxicity (DNT) has been assessed using experimental animals. Due to complexity of human brain development and species differences, alternative in vitro testing using human cells, such as human iPS cells (iPSCs), has been expected in terms of cost and time-consuming. In the present study, we examine the effects of a well-known anticancer agent 5-fluorouracil (5-FU) with potential DNT hazard (Mundy et al, 2015). We have focused on neural differentiation potency in iPSCs as an endpoint of DNT hazard evaluation. As a result, 5-FU reduced the expression of OTX2, a neurogenesis marker, in the neural induction. Since neural differentiation requires ATP as an energy, we examined the cellular ATP content. 5-FU decreased ATP levels in iPSCs. To understand the mechanisms of ATP reduction by 5-FU, we examined the effects of 5-FU on mitochondrial dynamics. 5-FU reduced mitochondrial fusion protein Mfn and induced mitochondrial fragmentation. Moreover, Mfn knockdown in iPSCs inhibited neural induction via OTX2 downregulation. These data suggest that 5-FU induces neurotoxicity via Mfn-mediated mitochondrial dysfunction in iPSCs. Thus, neural differentiation potency of iPSCs can be used for evaluation of drugs with DNT hazard.