<p>Background: Previous studies have suggested a protective role for Polyunsaturated Fatty Acids (PUFA) against cancer, cardiovascular, and other diseases. To provide new insights into the<em> in vivo</em> effects of PUFA on gene expression, the effects of dietary PUFA on <em>DNMT3b</em> and <em>PPAR&alpha;</em> gene expression and global DNA methylation were investigated in selected rat tissues.&nbsp;<br /> Methods: Thirty sprague-dawley rats were allotted into 3 dietary groups of ten animals each, received experimental diets containing PUFAs every day by gavages for 12 weeks as follows: control group fed a normal diet and water; n-3 PUFAs group received 300 <em>mg/kg/day</em> n-3 PUFAs supplementation; mixed-PUFAs group received 300 <em>mg/kg/day</em> of a mixture of n-3, -6, -9 PUFAs supplementations. The expressions of <em>DNMT3b</em> and <em>PPAR&alpha;</em> genes were quantitated using real-time RT-PCR. The genome-wide 5-methylcytosine contents in rat tissues were determined by ELISA method.&nbsp;<br /> Results: The average expression of the <em>DNMT3b </em>mRNA was 50% lower in the colon and liver of rats fed the n-3- or mixed-PUFAs supplemented diet than control group (p=0.00). However, <em>PPAR&alpha;</em> expression was significantly upregulated both in the colon and liver of PUFAs-supplemented rats (p&lt;0.001). No significant difference was observed in the blood, colon, and liver DNA methylation levels between PUFAs-supplemented and control animals.<br /> Conclusion: The results indicate that dietary PUFAs could modulate the expressions of <em>PPAR&alpha;</em> and <em>DNMT3b</em> genes in various rat tissues. The findings of this study provide additional insights into the in vivo mechanism of PUFA-mediated regulation of gene expression and could provide an opportunity to develop personalized diets for related disease control.</p>