Objective To observe the effect of Astragalus polysaccharides on the Ca²⁺ regulatory indexes of polyamine signaling pathway during the migration of rat intestinal epithelial cell line IEC-6 cells, so as to explore the mechanism by which Astragalus membranaceus promoting the repair of gastrointestinal mucosal injury. Methods The cell migration model was established by scratch method and the effect of Astragalus polysaccharide on cell migration was observed in calcium-free culture. The expression of transient receptor potential channel 1 (TRPC1), stromal interaction molecule (STIM)1 and STIM2 mRNA was determined by quantitative polymerase chain reaction. The distribution and expression of STIM1 protein were detected by immunofluorescence. The expression of TRPC1, STIM1 and STIM2 was detected by Western blotting. The expression of STIM1/TRPC1 and STIM1/STIM2 complexes was detected by immunoprecipitation. Results The promoting effect of Astragalus polysaccharides on cell migration was attenuated in calcium-free culture (removing the source of extracellular Ca²⁺ influx) (P<0.01). Astragalus polysaccharides had promoting effects on TRPC1 mRNA and protein expression (P<0.05 or P<0.01), and had reversal effects on TRPC1 mRNA and protein expression in difluoromethylornithine (DFMO) treated cells (P<0.01). Astragalus polysaccharide promoted the translocation of STIM1 to the plasma membrane and increased STIM1 protein expression, and reversed the translocation of STIM1 to the plasma membrane and the inhibition effect which was found in DFMO-treated cells. Astragalus polysaccharides increased the expression of STIM1 mRNA and protein and reversed the inhibition effects on STIM1 mRNA and protein expression induced by DFMO (P<0.05 or P<0.01). In addition, Astragalus polysaccharides reduced the expression of STIM2 mRNA and protein and reversed the increased expression of STIM2 mRNA and protein induced by DFMO (P<0.05 or P<0.01). Astragalus polysaccharides increased the expression of STIM1/TRPC1 complex and reversed the inhibition effect of DFMO on STIM1/TRPC1 complex expression, modulated the expression of STIM1/STIM2 by increasing STIM1 expression and reducing STIM2 expression, and antagonized the effect of DFMO on STIM1/STIM2 expression. Conclusion The role of Astragalus polysaccharides in promoting the migration of IEC-6 cells is related to its effect on the expression of Ca²⁺ regulatory protein and protein complex.