The objective of this paper was to study the relaxant effect mechanism of an estrone derivative EA303 on isolated jejunum smooth muscle of rabbits.The sample was prepared with rabbit isolated jejunum smooth muscle, and the effect of EA303 on the spontaneous contraction of jejunum smooth muscle was investigated.The effect of EA303 on β-receptor was studied by observing the difference of relaxing concentration-response curves of EA303 pre-contracted with KCl or acetylcholine chloride before and after incubation with propranolol (0.1 μmol/L-1).The influence of EA303 on the cumulative concentration-response curves of CaCl2 was studied, and verapamil was used to compare with EA303 as a noncompetitive inhibitor of Ca2+ channel.EA303 (10-100 μmol/L-1) significantly attenuated the tension and amplitude of spontaneous contraction of isolated jejunum smooth muscle.The relaxing concentration-response curves of EA303 pre-contracted by KCl or acetylcholine chloride significantly changed after incubation with propranolol (0.1 μmol/L-1).The relaxing effect of EA303 induced by KCl was inhibited, but increased induced by acetylcholine chloride.Both incubation with EA303 (1, 3 μmol/L-1) and verapamil (0.1 μmol/L-1) shifted the concentration-response curves of CaCl2 to the right, and the rightward shift of the concentration-response curves of CaCl2 was dose-dependent with the decrease of maximum responses.EA303, similar to verapamil, had significant effect on contraction of the first phase and second phase induced by acetylcholine chloride.The relaxant effect of EA303 on jejunum smooth muscle had relationship with β-receptor, and EA303 could block calcium channel and inhibit the release of intracellular calcium.