The 26S proteasome is the most complex protease that degrades most of intracellular proteins in eukaryotes. Although proteasome can be activated as the ubiquitinated substrates bind, this macromolecular machinery is dynamically checked through a variety of regulatory mechanisms. Three classes of deubiquitinating enzymes (USP14, RPN11, and UCH37) are associated with the deubiquitination reactions that regulates the proteolysis of human proteasome. Recent biochemical and structural studies have revealed that these enzymes have a dynamic impact on proteasome output depending on various cellular conditions, leading to different proteolysis outcomes. Among them, USP14 has been best known as a critical inhibitory component on the proteasome. USP14’s inhibitory activity relies on a unique multi-chain specific and en bloc cleavage mechanism. In contrast, USP14 was also reported to regulate opening of the core particle gate within the proteasome through a non-catalytic effect. By employing the fluorescent peptide substrates, here we show that the presence of USP14 can enhance the proteasome’s gate opening and peptide hydrolysis. A catalytically inactive USP14 C114A or Ub aldehyde treatment also stimulate peptide hydrolysis of proteasome. Furthermore, IU1 series, which are specific inhibitors of proteasome-bound form of USP14, significantly increased the proteasome’s peptidase activity in the presence of USP14 WT or C114A. Interestingly, IU2-6, which inhibits both proteasome-bound form and free form of USP14, does not induce gate opening. These results demonstrate that peptide entry is dependent on USP14’s allosteric regulation of proteasome, but not on the enzyme’s catalytic activity. This also indicates that distinct binding mode of USP14 inhibitors may exert differential effects on gate opening. Given that the deubiquitination activity of USP14 was known to rescue the substrate from proteasomal degradation, our data reveal another unexpected aspect of USP14 on proteolytic regulation. We further employed chemical inhibitors of different proteasome-associated deubiquitinating enzymes to further dissect the contribution of each deubiquitinating activity to the gate opening. Our results indicate that USP14 and IU1-specific USP14 inhibition, but not other proteasome-associated deubiquitinating activities, can influence the gate opening of the proteasome through allosteric regulation. We are currently investigating the hypothesis that proteasome-associated USP14 may communicate with proteasome’s catalytic activities through a long-range interaction in a bidirectional manner. Importantly, USP14 and its inhibitors could be applied to stimulate the proteasome activity in a dynamic context of proteasome and substrate, which might have potential therapeutic effects on clearance of disease-causing proteins.