Our objective was to identify the molecular mechanisms through which fish oil, a source of omega-3 polyunsaturated fatty acids (n-3 PUFA) in the hen diet influence adipose development in chick embryo. Broiler-breeder hens (n = 30/diet) were fed diets containing 2.3% of either fish oil or soybean oil (n-6 PUFA control) for four weeks. Fertilized eggs (n = 40/diet) from maternal fish oil (MFO) and soybean oil (MSO) were randomly collected and incubated to embryonic ages (E) 14, E16, and E20. Fatty acid profiles revealed that MFO significantly enriched yolk and subcutaneous adipose tissue in EPA and DHA. This difference was reflected in adipose tissue morphology, as histological analysis showed that MFO significantly reduced adipocyte size and increased the frequency of small adipocytes at each age examined. Preadipocytes isolated from E16 adipose tissue in MFO accumulated significantly less lipid than MSO controls, consistent with inhibition of fat accretion by fish oil. In addition, MFO preadipocytes exhibited significantly higher mitochondrial activity than MSO preadipocytes, attributed to their increased density, indicating improved oxidative metabolism capacity. Transcriptome profiling by RNA-seq in E16 adipose tissue revealed significant effects of MFO, with 210 upregulated and 113 downregulated genes compared to the MSO control. These genes were mainly enriched in pathways involved in lipid metabolism, fatty acid metabolism, and PPAR signaling pathway. Notably, MFO increased the expression of four members of the Iroquois homeobox genes, which have recently been shown to regulate adipogenesis in humans and rodents. The type of fatty acids in the hen diet also significantly influenced patterns of DNA methylation. A total of 17 differentially methylated regions were identified (FDR P < 0.1). Collectively, these results indicate that dietary programming by n-3 PUFA may reduce fat accretion after hatch through effects on transcriptomic and epigenetic mechanisms that originate in the embryo.