Activated skeletal muscle proteolysis in catabolic states has been linked to an upregulation of the ATP-ubiquitin-dependent proteolytic system. Previous studies suggested that the N-end rule pathway is primarily responsible for the bulk of skeletal muscle proteolysis. The activity of this pathway is dependent on the 14-kDa ubiquitin-conjugating enzyme E2_14k (HR6B) and the ubiquitin protein ligase Ubr1. To address the requirement of E2_14k in muscle proteolysis, we examined muscle protein metabolism in wild-type (WT) mice and mice lacking the E2_14k gene (KO) in fed and fasted (48 h) states. Baseline body weight, muscle mass, and protein content were similar, and these parameters decreased similarly upon fasting in the two genotypes. There were also no effects of genotype on the rate of proteolysis in soleus muscle. The fasting-induced increase in the amount of ubiquitinated proteins was the same in WT and KO mice. The absence of any significant effect of loss of E2_14k function was not due to a compensatory induction of the closely related isoform HR6A. Total intracellular concentration of E2_14k and HR6A in the WT mice was 290 ± 40 nM, but the level in the KO mice (reflecting the level of HR6A) was 110 ± 9 nM. This value is about threefold the apparent Michaelis-Menten constant (K_m) of E2_14k (~40 nM) for stimulating conjugation in muscle extracts. Because the HR6A isoform has a K_m of 16 nM for stimulating conjugation, the HR6A levels in the muscles of KO mice appear sufficient for supporting conjugation mediated by this pathway during fasting.