Castration increases fat deposition, improving beef quality in cattle. Here, the steer group exhibited a significantly higher intramuscular fat (IMF) content than the bull group. To determine the potential roles of microRNAs (miRNAs) in castration-induced fat deposition, differential expression patterns of miRNA in liver tissue were investigated in bulls and steers. A total of 7,827,294 clean reads were obtained from the bull liver library, and 8,312,483 were obtained from the steer liver library; 452 conserved bovine miRNAs and 20 novel miRNAs were identified. The results showed that the expression profiles of miRNA in liver tissue were changed by castration, and 12 miRNAs that were differentially expressed between bulls and steers were identified. Their target genes were majorly involved in the metabolic, PI3K-Akt, and MAPK signaling pathways. Furthermore, six differentially expressed miRNAs were validated by quantitative real-time PCR, and luciferase reporter assays verified that calcium-sensing receptor (CASR) was the direct target of miR-27a-5p. Meantime, we found that the expression level of CASR was significantly higher in steers than in bulls, and revealed that CASR gene silencing in bovine hepatocytes significantly inhibited triacylglycerol (TAG) accumulation and reduced secretion of very low density lipoprotein (VLDL). These results obtained in the liver indicate that miR-27a-5p may increase fat deposition partly by targeting CASR in steers.