This study aims to investigate the global profiling of genes and miRNAs expression to explore the regulatory effects of eicosapentaenoic acid (EPA) in visceral adipose tissue (VAT) of obese mice. We used male mice, fed either a high-fat diet (HF) or HF supplemented with EPA (HF-EPA), for 11 weeks. RNA, and small RNA profiling, were performed by RNAseq analysis. We conducted analyses using Ingenuity Pathway Analysis software (IPA®) and validated candidate genes and miRNAs related to lipid mediators and inflammatory pathways using qRT-PCR. We identified 153 genes differentially downregulated, and 62 microRNAs differentially expressed in VAT from HF-EPA compared to HF. Genes with a positive association with inflammation, chemotaxis, insulin resistance, and inflammatory cell death, such as Irf5, Alox5ap, Tlrs, Cd84, Ccr5, Ccl9, and Casp1, were downregulated by EPA. Moreover, EPA significantly reduced LTB4 levels, a lipid mediator with a central role in inflammation and insulin resistance in obesity. The pathways and mRNA/microRNA interactions identified in our study corroborated with data validated for inflammatory genes and miRNAs. Together, our results identified key VAT inflammatory targets and pathways, which are regulated by EPA. These targets merit further investigation to better understand the protective mechanisms of EPA in obesity-associated inflammation.