MicroRNAs (miRNAs) are small regulatory molecules (~22 nt) which cause post-transcriptional gene silencing. Our objective was to examine the effect of overfeeding a moderate-energy diet on miRNA expression in mesenteric adipose tissues of dry non-pregnant cows. Fourteen Holstein cows were assigned to treatments in a randomized complete block design. All cows were fed individually a control diet (CON; NEL = 1.3 Mcal/kg) to meet 100% of NRC requirements for 3 wk, after which half of the cows were assigned to a moderate-energy diet (OVE; NE_L = 1.6 Mcal/kg) and half of the cows continued on CON for 6 wk. The OVE diet was fed ad libitum and resulted in cows consuming energy at ~180% of NRC. CON cows were fed to consume only to 100% of NRC. The BW was measured from wk −3 to 6 wk. Cows were slaughtered after 6 wk, and samples of mesenteric fat collected for total RNA plus miRNA extraction, and miRNA sequencing. The differential expression of miRNAs in OVE vs. CON was assessed using EdgeR with an FDR adjusted P-value (P < 0.05). The analysis of predicted miRNA target genes was performed by estimating a Total Context+ Value (TC+V ≤ −0.7) using the TargetScan database. OVE cows had greater (P < 0.001) BW at slaughter (758 vs. 693 kg). Fourteen miRNAs were differentially expressed (P < 0.05); the OVE cows had a greater expression of miRNAs bta-miR-378, 22–3p, 107, 103, 149–5p, 378c, 30c, 1307 and 6529a, whereas, the bta-miR-199a-5p, 205, 339b, 339a and bta-miR-27a-3p were upregulated in CON cows. The TargetScan analysis predicted the transcriptional change of 47 genes related to cellular ion transport and binding (e.g., Na⁺/K+ and calcium), fat metabolism (PPARGC1B) and feeding behavior (GPR178). Overfeeding energy did not alter insulin sensitivity but seemed to affect adipogenesis and fatty acid metabolism in mesenteric adipose tissue. In OVE cows, PPARG was the main target gene triggered by bta-miR-30c upregulation. The results suggest that miRNAs can respond to dietary energy level and likely play a role in regulating adipose tissue metabolism and energy expenditure.