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Few studies have been conducted regarding the biological function and regulation role of gga-miR-221-5p in the liver. We compared the conservation of miR-221-5p among species and investigated the expression pattern of gga-miR-221-5p, validating the direct target genes of gga-miR-221-5p by dual luciferase reporter assay, the biological function of gga-miR-221-5p in the liver was studied by gga-miR-221-5p overexpression and inhibition. Furthermore, we explored the regulation of gga-miR-221-5p and its target genes by treatment with estrogen and estrogen antagonists in vivo and in vitro. The results showed that miR-221-5p was highly conserved among species, expressed in all tested tissues and significantly downregulated in peak-laying hen liver compared to pre-laying hen liver. Gga-miR-221-5p could directly target the expression of elongase of very long chain fatty acids 6 (<i>ELOVL6)</i> and squalene epoxidase (<i>SQLE</i>) genes to affect triglyceride and total cholesterol content in the liver. 17&#946;-estradiol could significantly inhibit the expression of gga-miR-221-5p but promote the expression of <i>ELOVL6</i> and <i>SQLE</i> genes. In conclusion, the highly conservative gga-miR-221-5p could directly target <i>ELOVL6</i> and <i>SQLE</i> mRNAs to affect the level of intracellular triglyceride and total cholesterol. Meanwhile, 17&#946;-estradiol could repress the expression of gga-miR-221-5p but increase the expression of <i>ELOVL6</i> and <i>SQLE</i>, therefore promoting the synthesis of intracellular triglyceride and cholesterol levels in the liver of egg-laying chicken.