Abstract: To explore the role of DNA methylation in the effects of testosterone deficiency induced obesity in miniature pigs fed on a high-fat diet（HFD）, the study employed methylated DNA immunoprecipitation sequencing（MeDIP-Seq） technology to examine DNA methylation alterations in the visceral adipose tissue（VAT） of HFD-fed intact male pigs（SHAM）, castrated male pigs（CAS） and castrated male pigs with testosterone treatment（CAS+T）. Differentially methylated genes（DMGs） were screened out and used for functional enrichment analysis. RT-qPCR was used to detect the expression of DMGs. The results showed that the genome distribution of DNA methylation was similar among SHAM, CAS and CAS+T, with a relatively higher methylation level in the gene body region than that in the 3’UTR and 5’UTR. Furthermore, 2 839 and 2 510 DMGs were obtained from CAS vs. SHAM and CAS vs.CAS+T, respectively. These DMGs were mainly enriched in adipocytokine signaling pathway, antigen processing and presentation, fatty acid metabolism and amino acid metabolism. Testosterone deficiency caused the increase of DNA methylation in LEP and NCF1 intron and in SLC27 A1 promoter. Levels of LEP and NCF1 m RNA expression were positively correlated with their methylation, while the SLC27 A1 mRNA level was negatively correlated with its methylation. Our study speculated that testosterone deficiency might regulate fat deposition and obesity through affecting DNA methylation of genes involved in lipid metabolism and inflammatory response related multiple pathways.