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MiRNAs are vital regulators and play a major role in cell differentiation, biological development, and disease occurrence. In recent years, many studies have found that miRNAs are involved in the proliferation and differentiation of adipocytes. The objective of this study was to evaluate the effect of miR-27a and its target gene <i>CPT1B</i> on ovine preadipocytes differentiation in Small-tailed Han sheep (<i>Ovis aries</i>). Down-regulation of miR-27a significantly promoted the production of lipid droplets, while overexpression of miR-27a led to a reduction in lipid droplet production. In addition, inhibition of miR-27a led to a significant increase in the expression of genes involved in lipid synthesis, including PPAR γ, SCD, LPL, and FABP4. Target Scan software predicted that <i>CPT1B</i> is a new potential target gene of miR-27a. Further experiments revealed that <i>CPT1B</i> gene expression and protein levels were negatively correlated with miR-27a expression. Overexpression of miR-27a led to a significant decrease in <i>CPT1B</i> mRNA levels and inhibited the accumulation of lipid droplets and vice versa. Moreover, overexpression of <i>CPT1B</i> promoted the synthesis of lipid droplets in ovine preadipocytes. Furthermore, luciferase reporter assays confirmed <i>CPT1B</i> to be a miR-27a direct target gene. This study confirmed that miR-27a increases the expression of genes related to lipid synthesis in ovine preadipocytes by targeting <i>CPT1B</i>, thereby promoting the synthesis of lipid droplets. The results of this study can be used to be exploited in devising novel approaches for improving the IMF content of sheep.