Transgene silencing is a common phenomenon in higher plants and microalgae. In the present study, we analyzed transgene silencing in the macroalga Pyropia yezoensis. This is the first report of transgene silencing in macroalgae. Transgenic lines were generated by transformation with the PyGUS gene and a hygromycin resistance gene (PtAph7-1). Histochemical GUS staining detected PyGUS expression in only three out of 12 lines. Southern and northern blotting, polymerase chain reaction (PCR), and reverse transcription (RT)-PCR analyses indicated that the PyGUS gene was silenced in the other 9 lines. Interestingly, PyGUS gene silencing in transgenic line 7 was repeatedly initiated at a later stage, in the developmental process from monospore to thallus through asexual reproduction. We identified genes encoding Argonaute and Dicer-like protein, the major components of the RNA-silencing pathway, in the genome and transcripts of P. yezoensis. Interestingly, we found the DDA motif in the PIWI domain of the AGO, suggesting that translational repression may be the major gene-silencing pathway in P. yezoensis. In this study, the observation of PyGUS gene silencing and identification of RNA-silencing components indicate that the gene ( transgene) silencing machinery functions actively in the macroalga P. yezoensis. These results will be useful to study the regulation of gene expression and RNA-silencing mechanisms in Pyropia species and related macroalgae.