Phytanic acid is a branched fatty acid that is a metabolic intermediate of chlorophyll. In this study, the effects of phytanic acid on Histone deacetylase (Hdac) activity were examined in an in vitro enzyme assay and in neuronal Neuro2a cells. Several fatty acids have been shown to be Hdac inhibitors, but phytanic acid enhanced the enzyme activity in vitro. In Neuro2a cells, phytanic acid significantly reduced histone acetylation and induced cell death, which was inhibited by an Hdac inhibitor, sodium butyrate. Theophylline, a common Hdac activator, had a similar effect on Neuro2a cell viability, and this effect was also inhibited by sodium butyrate. Phytanic acid decreased the level of intracellular active mitochondria, while butyrate increased this level. The cytotoxic effect of phytanic acid was also abolished by a caspase-9 inhibitor. Apicidin, a Hdac2- and 3-specific inhibitor, reduced the cellular toxicity, which suggests that the toxicity of phytanic acid depends on activation of the Hdac2 and 3 subtypes. Overall, these results show that phytanic acid induces mitochondrial abnormality and cell death via activation of Hdac2, 3 in Neuro2a cells. This effect of Hdac activation by phytanic acid may produce neuronal damage in Refsum disease and other peroxisomal disorders, which is caused by accumulation of phytanic acid.