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DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific <i>piwi</i> gene expression during spermatogenesis of Japanese flounder (<i>Paralichthys olivaceus</i>), the expression profiles of <i>piwil1</i> (<i>piwi-like 1</i>) and <i>piwil2</i> (<i>piwi-like 2</i>) genes in the gonads of female, male, and sex-reversed pseudo-male <i>P. olivaceus</i> were analyzed, and the dynamic of DNA methylation was investigated. As a result, <i>piwil1</i> and <i>piwil2</i> genes were highly expressed in the testis of both male and pseudo-male <i>P. olivaceus</i>, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both <i>piwil1</i> and <i>piwil2</i> were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of <i>piwi</i> genes during spermatogenesis. There was also sperm quality variation among male <i>P. olivaceus</i>, and the sperm curvilinear velocity was positively correlated with the expression of both <i>piwil1</i> and <i>piwil2</i> genes. These results indicated that the DNA methylation in <i>piwil1</i> and <i>piwil2</i> promoter regions may affect the initiation of <i>piwi</i> gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in <i>P. olivaceus</i>.