This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×‐RDA), or 2.4 (3×‐RDA) g · kg^–1·· d^–1 for 31 d. A 10‐d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)‐mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence‐based assays, immunoblot analysis, and real‐time qRT‐PCR In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up‐regulation of UPS‐associated gene expression, as mRNA expression of the atrogenes muscle RING finger‐1 (MuRF1) and atrogin‐1 were 1.2‐ and 1.3‐fold higher (P<0.05) for ED than for WM. However, mixed‐meal consumption attenuated UPS‐mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin‐1 mRNA expression during ED, consuming a protein‐containing mixed meal attenuates Ub‐mediated proteolysis.—Carbone, J. W., Margolis, L. M., McClung, J. P., Cao, J. J., Murphy, N. E., Sauter, E. R., Combs, G. F., Jr., Young, A. J., Pasiakos, S. M., Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis. FASEB J. 27, 5104–5111 (2013). www.fasebj.org