Pufferfish saxitoxin and tetrodotoxin binding proteins (PSTBPs) play an important role in the toxification of certain species of pufferfish. Recombinant Takifugu rubripes PSTBP1 (rTrub.PSTBP1) is reported to bind to tributyltin, and so it has been suggested that rTrub.PSTBP1 may reduce the toxicity of tributyltin. However, the role of PSTBP1 in vivo remains to be elucidated. Here, we established a transgenic medaka line showing whole-body Renilla reniformis green fluorescent protein and Trub.PSTBP1 expression, as confirmed by real-time polymerase chain reaction and mRNA-Seq analysis. mRNA-Seq analysis also showed that cytochrome P450 superfamily genes and the gene encoding ATP-binding cassette sub-family G member 2 were highly expressed in the transgenic medaka. Using embryos of the transgenic medaka line, we conducted an in ovo nanoinjection test to examine the effect of Trub.PSTBP1 in vivo, and obtained data suggesting that Trub.PSTBP1 expression may have reduced the toxicity of tributyltin in our transgenic medaka line. Our findings will be useful for future functional analyses of Trub.PSTBP1.