Abstract: To investigate whether the intestinal flora culture supernatant of mice consuming polysaccharides from Ziziphus jujuba Mill var. spinosa seeds (ZSSP) could affect the expression levels of efflux transporters, tight junction proteins and phase Ⅱ metabolizing enzymes in Caco-2 cells, twenty C57BL/6 mice were randomly divided into a control group and a ZSSP group, which were administrated with ultra-pure water and ZSSP solution (100 mg/kg·d^-1) for 14 d, respectively. Mice feces were collected to prepare intestinal flora culture supernatant. The effect of intestinal flora culture supernatant on Caco-2 cell viability was measured by methyl thiazolyl tetrazolium (MTT) method. Western blot was used to detect the expression levels of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), occludin, and claudin-1 in Caco-2 cells. Enzyme linked immunosorbent assay (ELISA) kits were used to determine the content of sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT). The results showed that, the intestinal flora culture supernatant of the ZSSP group could significantly reduce the expression of efflux transporters (P-gp and MRP2) and significantly increase the expression of UGT in Caco-2 cells (P<0.05). However, it had no effect on tight junction proteins (occludin and claudin-1) and SULT (P>0.05). It can be seen that the intestinal flora culture supernatant of mice consuming ZSSP can regulate the expression level of efflux transporter protein and phase II metabolic enzymes in Caco-2 cells.