Objective The purpose of this study is to understand the influence of Fkbp51 gene knockout on alternative splicing of mRNA in liver tissues. Methods mRNAs of liver from both Fkbp51 knockout(KO) and wild type(WT) mice were isolated. mRNA expression profiling was performed using RNA-seq reads. The mRNA reads produced from RNA-seq was analysed by TopHat for alternative splicing. Exon skipping and intron retetion were identified according to alignment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed using online tools DAVID. All genes which had differently expressed alternative splicing between WT and KO were annotated using NCBI database. Results (1) Fkbp51 KO can introduce new mRNA alternative splicing; (2) Fkbp51 KO also cause mRNA alternative splicing expression change; (3) According to GO and KEGG analysis, it was found that the mRNA alternative splicing changes mainly enriched in fat metabolism, immune, bile acid secretion, and PPAR signaling pathway etc. (4) The genes which have different intron retetion events are mainly involved in regulation of actin cytoskeleton organization and amino acids metabolism. Conclusions Fkbp51 gene knockout can cause the change of mRNA alternative splicing, and thereby affect the metabolism of liver in mice.