Objective The aim of this study was to explore the effect of testosterone deficiency on mRNA alternative splicing in the liver of miniature pigs fed a high?fat and high?cholesterol diet (HFC). Methods RNA?Seqanalysis was employed to characterize the global transcriptome changes in the liver of intact male pigs (SHAM), castrated male pigs (CAS) and castrated male pigs with testosterone treatment (CAS + T). TopHat was used to identify the testosterone?regulated alternative splicing ( AS) events. After being annotated, Gene Ontology ( GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further performed for the identified common differentially expressed AS genes between the three groups. Results 1) Castration and testosterone treatment introduced new mRNA alternative splicing events in the liver of miniature pigs fed a HFC. 2) There were 113 common differentially expressed AS genes between SHAM vs. CAS and CAS + T vs. CAS groups, which including many lipid and glucose metabolism?related genes, i. e. , AGPAT6, NR1H4, PPARD and GK. 3) According to the GO and KEGG analysis, the 113 genes mainly enriched in fatty acid metabolic process, glycerolipid metabolism, responsed to glucose and adipocytokine signaling pathway. Conclusions Testosterone deficiency affects mRNA alternative splicing in the liver of miniature pigs fed a HFC. Moreover, testosterone deficiency may promote diet?induced fatty liver disease through regulating the alternative splicing of genes involved in lipid and glucose metabolism. Further study is needed to explore the relationship between testosterone and the AS genes and their new transcripts.