The intestinal epithelium is known as an important barrier to protect the body from harmful pathogens or toxic substance that may induce intestinal barrier injury. The aim of this study was to investigate the effects of polysaccharide from the seeds of Plantago asiatica L. (PLP) on nonylphenol (NP) induced intestinal barrier injury in vitro. Caco-2 cells were pretreated with PLP, or co-cultured with PLP and NP simultaneously, and cytotoxicity, LDH leakage, transepithelial electrical resistance (TEER), FITC-dextran flux and tight junction (TJ) proteins were conducted to evaluate the intestinal barrier function. The results suggested that PLP pretreatment or co-culture with NP could significantly attenuated NP induced Caco-2 cytotoxicity, suppressed LDH release, restored the TEER value and paracellular permeability of Caco-2 monolayers, which were attributed to enhancing the TJ protein expressions. In addition, PLP co-cultured with NP possessed better protective effects against NP induced cytotoxicity. This study indicated that PLP assuaged NP induced intestinal barrier injury by increasing TJ, and threw light on the development of a dietary supplementation for preventing exogenous toxic substances induced intestinal barrier injury or improving intestinal TJ barrier function.