Biological barrier plays a crucial role for organisms against the possible toxicity from engineered nanomaterials (ENMs). Graphene oxide (GO) has been proven to cause potential toxicity on organisms. However, the molecular mechanisms for intestinal barrier of animals against GO toxicity are largely unclear. Using in vivo assay system of Caenorhabditis elegans, we found that mutation of genes encoding core p38 mitogen-activated protein kinase (MAPK) signaling pathway caused susceptible property to GO toxicity and enhanced translocation of GO into the body of nematodes. Genetic assays indicated that SKN-1/Nrf functioned downstream of p38 MAPK signaling pathway to regulate GO toxicity and translocation. Transcription factor of SKN-1 could regulate GO toxicity and translocation at least through function of its targeted gene of gst-4 encoding one of phase II detoxification proteins. Moreover, intestine-specific RNA interference (RNAi) assay demonstrated that the p38 MAPK-SKN-1/Nrf signaling cascade could function in intestine to regulate GO toxicity and intestinal permeability in GO exposed nematodes. Therefore, p38 MAPK-SKN-1/Nrf signaling cascade may act as an important molecular basis for intestinal barrier against GO toxicity in organisms. Exposure to GO induced significantly increased expression of genes encoding p38 MAPK-SKN-1/Nrf signaling cascade, which further implies that the identified p38 MAPK-SKN-1/Nrf signaling cascade may encode a protection mechanism for nematodes in intestine to be against GO toxicity.