ABSTRACT: Testosterone deficiency causes fat deposit, particularly in the visceral region, and its replacement might reverse fat accumulation, but the underlying mechanisms of such processes are largely unclear. To gain insights into the genome-wide role of testosterone on visceral adipose tissue (VAT), RNA-Seq was used to investigate testosterone deficiency induced changes of VAT in miniature pigs fed a high-fat and high-cholesterol (HFC) diet among intact male pigs (IM), castrated male pigs (CM), and castrated male pigs with testosterone replacement (CMT) treatments. The results showed that testosterone deficiency induced VAT deposit and increased serum leptin level. Moreover, a total of 1,732 differentially expressed genes (DEGs) were identified between different two groups. Compared with gene expression profiles in IM and CMT pigs, upregulated genes in CM pigs, i.e., LOC100520753 (CD68), LCN2, EMR1, NCF1 (p47phox), and NOX2 (GP91-PHOX), were mainly enriched in inflammatory response, oxidation-reduction process, and response to oxidative stress, while downregulated genes in CM pigs, i.e., DIO3, PCK1, and ABHD5, mainly focused on small molecule metabolic process. Taken together, our study provides a novel genome-wide view on the role of testosterone on VAT deposit under HFC diet, thus improving our understanding of the molecular mechanisms involved in VAT changes induced by testosterone deficiency.