Epidermal growth factor (EGF) has many important biological functions. It plays an important role in regulating the growth, survival, migration, apoptosis, proliferation, and differentiation of intestinal tissues and cells. However, until now, the effect of inflammation on the biological activity of EGF in intestinal cells or tissues is still unclear. For this reason, in the current research, we have conducted a detailed study on this issue. Using the rat small intestinal crypt epithelial cell line (IEC6) was used as an in vitro model, and Confocal laser scanning microscope (CLSM), Flow cytometry (FCM), Indirect immunofluorescence assay (IFA), Western-blotting (WB), and Quantitative real-time RT-PCR (QRT-PCR) methods were used to explore the effects of inflammation on EGF/EGFR biological activity and signal transduction profiles. We found that the EGF/EGFR nuclear signal almost disappeared in the inflammatory state, and the phosphorylation levels of EGFR, AKT, and STAT3 were all significantly down-regulated. In addition, we also studied the effect of β-carotene on the biological activity of EGF, and found that when cells were pretreated with β-carotene, the cellular behavior, biological activity, and nuclear signal of EGF/EGFR under inflammation stimulation were partially restored. In summary, the current study shows that inflammation can disrupt EGF/EGFR-mediated signaling in IEC6 cells, suggesting that inflammation negatively regulates the biological activity of EGF/EGFR. Furthermore, we found that β-carotene not only attenuated lipopolysaccharide (LPS)-induced inflammation but also partially restored the biological activity of EGF in IEC6 cells, laying a solid foundation for studying the biological functions of EGF and β-carotene.