Activated skeletal muscle proteolysis in catabolic states has been linked to an up-regulation 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 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 (48h) states. Baseline body weights, 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 (reflecting the level of HR6A) was 110 ± 9 nM. This value is about three fold the apparent Km of E2_14k (~40nM) for stimulating conjugation in muscle extracts. Since the HR6A isoform has a Km 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.