The objective of the present investigation was to determine whether energy restriction (ER) influences expression of skeletal muscle-specific microRNA (miRNA) in circulation (c-myomiR) and whether changes in c-myomiR are associated with rates of whole body protein synthesis. Sixteen older (64 ± 2 yr) overweight (28.5 ± 1.2 kg/m²) men enrolled in this 35-day controlled feeding trial. A 7-day weight maintenance (WM) period was followed by 28 days of 30% ER. Whole body protein turnover was determined from [¹⁵N]glycine enrichments in 24-h urine collections, and c-myomiR (miR-1-3p, miR-133a-3p, miR-133b, and miR-206) expression was assessed from serum samples by RT-quantitative PCR upon completion of the WM and ER periods. Participants lost 4.4 ± 0.3 kg body mass during ER ( P < 0.05). After 28 days of ER, miR-133a and miR-133b expression was upregulated ( P < 0.05) compared with WM. When all four c-myomiR were grouped as c-myomiR score (sum of the median fold change of all myomiR), overall expression of c-myomiR was higher ( P < 0.05) at ER than WM. Backward linear regression analysis of whole body protein synthesis and breakdown and carbohydrate, fat, and protein oxidation determined protein synthesis to be the strongest predictor of c-myomiR score. An inverse association ( P < 0.05) was observed with ER c-myomiR score and whole body protein synthesis ( r = −0.729, r² = −0.530). Findings from the present investigation provide evidence that upregulation of c-myomiR expression profiles in response to short-term ER is associated with lower rates of whole body protein synthesis.