Tributyltin (TBT), a common organotin environmental pollutant, has been widely used as a component of marine antifouling paints. We previously reported that exposure to TBT inhibits the expression and DNA binding of nuclear respiratory factor-1 (NRF-1) and causes neurotoxicity. In the present study, we focused on the epigenetic effects of TBT and investigated whether TBT decreases NRF-1 expression via epigenetic modifications in SH-SY5Y human neuroblastoma cells. First, we found that exposure to 300 nM TBT decreases NRF-1 expression. We examined epigenetic changes induced by TBT, and showed that TBT causes hypermethylation of the NRF-1 promoter region, increases the amount of methyl-CpG-binding protein 2 (MeCP2) bound to the NRF-1 promoter, and alters the expression of DNA methyltransferases and ten-eleven translocation (TET) demethylation enzymes. These results suggest that epigenetic changes play an important role in regulation of NRF-1 expression. Next, we investigated effect of NRF-1 expression decrease on cells, and TBT reduces mitochondrial membrane potential and overexpression of NRF-1 rescued this reduction in membrane potential. Thus, we suggested that NRF-1 is important for maintaining mitochondrial membrane potential. Our study indicates that TBT causes epigenetic changes such as hypermethylation, which increases recruitment of MeCP2 to the NRF-1 promoter and probably lead to decreased of NRF-1 expression and mitochondrial membrane potential. Therefore, this research provides new evidence of the epigenetic action caused by organotin.