P-Glycoprotein (P-gp), one of the drug efflux pumps, is expressed in some tissues and may affect the pharmacokinetics of its substrates. We have previously reported that a decrease of intestinal P-gp expression affects the pharmacokinetics of orally-administered P-gp substrate drugs in a streptozotocin (STZ)-induced type 1 diabetic mice model. Although we have found the participation of nitric oxide synthase (NOS) activation as a mechanism of the decrease in intestinal P-gp expression under diabetic conditions, more detailed mechanisms other than NOS remain unknown. Here, we studied the involvement of the ubiquitin-proteasome system in the mechanism of the decrease in intestinal P-gp expression under diabetic conditions. Nine days after STZ administration (diabetic condition), ubiquitination levels of ileal P-gp were significantly increased, accompanied by an decrease of intestinal P-gp protein expression levels. Furthermore, treatment with an NO donor could increase the intestinal ubiquitinated P-gp levels. On the other hand, activity of 26S proteasome, an important enzyme in ubiquitin-proteasome system, did not change, suggesting the first step of the system (i.e., ubiquitination) but not the second step (i.e., degradation)-specific up-regulation under diabetic conditions. Our results reveal the participation of the acceleration of the ubiquitin-preotasome system by NO in the decrease of intestinal P-gp expression levels under diabetic conditions.