There was no significant difference in the composition and content of fatty acids in eggs among different breeds initially, but following the supplementation of flaxseed oil, Dwarf Layer were observed to deposit more n-3 polyunsaturated fatty acid (PUFA) in eggs. Currently, there is limited research on the mechanisms underlying the differences in egg composition among different breeds. Therefore, in this study, 150 twenty-four-wk-old hens of each breed, including the Dwarf Layer and White Leghorn, were fed either a basal diet or a diet supplemented with 2.5% flaxseed oil. After 28 d, eggs and liver samples were collected to determine fatty acid composition, and serum, liver, intestine, and follicles were collected for subsequent biochemical, intestinal morphology, and lipid metabolism-related genes expression analysis. Duodenal contents were collected for microbial analysis. The results showed that there was no significant difference in the content and deposition efficiency of total n-3 PUFA in the liver of the 2 breeds, but the content and deposition efficiency of total n-3 PUFA in the egg of Dwarf Layer were significantly higher than those of White Leghorn after feeding flaxseed oil. Flaxseed oil and breeds did not have significant effects on cholesterol (CHO), free fatty acids (NEFA), low-density lipoprotein (LDL), and estrogen (E 2 ) levels. After feeding with flaxseed oil, the villus height and the villus-to-crypt ratio in both breeds were increased and duodenal crypt depth was decreased. The villus-to-crypt ratio (4.78 vs. 3.60) in the duodenum of Dwarf Layer was significantly higher than that in White Leghorn after feeding with flaxseed oil. Flaxseed oil can impact the gut microbiota in the duodenum and reduce the microbiota associated with fatty acid breakdown, such as Romboutsia, Subdolibranulum, Lachnochlostridium, and Clostridium. This may mean that less ALA can be decomposed and more ALA can be absorbed into the body. Additionally, after feeding flaxseed oil, the mRNA levels of elongation enzymes 5 (ELOVL5), fatty acid desaturase 1 (FADS1), and fatty acid transporter 1 (FATP1) in the liver of Dwarf Layer were significantly higher than those in White Leghorn, while the mRNA levels of peroxisome proliferator-activated receptor alpha (PPAR), carnitine palmitoyl transferase 1 (CPT1), Acyl CoA oxidase 1 (ACOX1), and Acyl-CoA synthetase (ACSL) were significantly lower than those in White Leghorn. The mRNA level of FABP1 in the duodenum of Dwarf Layer was significantly higher than that of White Leghorn, while the mRNA level of FATP1 was significantly lower than that of White Leghorn. The protein levels of ELOVL5 in the liver of Dwarf Layer and very low-density lipoprotein receptor (VLDLR) in the follicles were significantly higher than those of White Leghorn. In summary, after feeding flaxseed oil, the higher ratio of villus height to crypt depth in Dwarf Layer allows more α-linolenic acid (ALA) to be absorbed into the body. The higher mRNA expression of FADS1, ELOVL5, and FATP1, as well as the higher protein expression of ELOVL5 in the liver of Dwarf Layer enhance the conversion of ALA into DHA. The higher protein expression of VLDLR in follicles of Dwarf Layer allows more n-3 PUFA to deposit in the follicles. These combined factors contribute to the Dwarf Layer's ability to deposit higher levels of n-3 PUFA in eggs, as well as improving the deposition efficiency of n-3 PUFA., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)