The pufferfish accumulates neurotoxic tetrodotoxin in its body and inflates by filling its stomach with water. These traits are unique to this species, and may be a result of adaptation post-divergence of Tetraodontidae. However, evolution of the protein-coding genes in the pufferfish has not yet been well elucidated. Detection of positive selection on these genes can help us understand the mechanisms associated with functional evolution. We downloaded well-annotated gene information of two pufferfish species, Takifugu rubripes and Tetraodon nigroviridis, from the public ENSEMBL database. In order to detect selective pressure on protein-coding sequences, we performed dN/dS estimation using codeml within the PAML software package. We selected one to one orthologous genes among seven fish species (Gasterosteus aculeatus, Oryzias latipes, Poecilia formosa, Takifugu rubripes, Tetraodon nigroviridis, and Xiphophorus maculatus). Results of dN/dS analysis on orthologous genes indicate that pufferfish showed high non-synonymous substitution rate for positively selected genes, and the evolutionary rate was faster during the diversification of two pufferfishes after divergence. Additionally, a candidate mechanism for regulation of neuro-toxicity of tetrodotoxin was identified from functional annotation of positively selected genes. These results support positive selection on protein-coding genes of the pufferfish with the acquisition of specific phenotypic traits.