The aim of this study was to investigate the effect of epidermal growth factor (EGF) on phosphate absorption across intestinal mucosa of hens using the Ussing chamber system. First, four Loman laying hens with similar weight (1.25±0.13 kg) were sacrificed to detect the lactate dehydrogenase (LDH) activity of isolated small intestine at time on 0 min, 30 min, 60 min, 90 min and 120 min, to provide a appropriate operation time for Ussing chamber test. Then, a total of 20 Loman laying hens with similar weight (1.25±0.13 kg) were divided into 1 of 5 treatments: i) control group (0 ng/mL EGF), ii) 50 ng/mL EGF group, iii) 100 ng/mL EGF group, iv) 150 ng/mL EGF group, and v) 200 ng/mL EGF group for using chamber test. The results showed that (i) the LDH activity of isolated intestine increased (P < 0.05) sharply during 30 min, at 60 min, the LDH activity of isolated intestines reached a high level; (ii) EGF increased (P < 0.05) the transepithelial electrical resistance (TEER, Ω.cm2) significantly; (iii) EGF inhibited (P < 0.05) the phosphate diffusion across intestinal mucosa significantly. According to the results, the Ussing chamber system can use to evaluate phosphate diffusion across intestinal mucosa, and EGF may inhibit the absorption of phosphorus.