Herb-drug interactions are important safety concerns in clinical practice. The interactions occur firstly in the intestinal absorption for orally administered drugs. Aspirin and Panax notoginseng saponins (PNS)-based drugs are often combined in China to prevent larger-artery atherosclerosis. Here, we aimed to characterize the aspirin transport across Caco-2 cell monolayers, a model of the intestinal absorption, and further to evaluate the influence of PNS on aspirin hydrolysis and the relating mechanisms. Transcellular transport of aspirin and the influence of PNS were explored using Caco-2 cell monolayers. The protein expression of human carboxylesterase 1 (hCE1) and hCE2 in Caco-2 cells after PNS treatment was analyzed by ELISA, and the mRNA level were determined by qRT-PCR. In the study, Caco-2 cells showed high level of hydrolase activity, and most aspirin was hydrolyzed inside the cells during the transport process. Interestingly, PNS were demonstrated to inhibit the esterase activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of hCE1 and hCE2, whereas exhibited minor effect on the mRNA expression. These results indicated that oral administration of PNS-based drugs might inhibit the hydrolysis of aspirin during intestinal absorption thus promoting its bioavailability.