In order to investigate the feasibility of electron beam induced mutation on Gladiolus and the effects of electron beam with different doses on pollen traits, M1 generation pollen traits were studied by use of 3MeV electron beams with seven doses of 0Gy, 40Gy, 80Gy, 120Gy, 160Gy, 200Gy, 240Gy. It has been shown that the sterile pollen rate of M1 generation is higher than that of control. At low dose there had no obvious effects on the viability and germination rate of M1 generation pollen. With the increase of irradiation dose, the viability and germination rate of pollen was decreaced and the difference had statistical significance (,P,<,0.05). Comparing (SEM) photograph of the pollen shapes of M1 generation with control, within a certain doses level, mutagenic effects on pollen increased with the dose increasing in the angle of polynology. Variations of mutagenic effect extent were found at the different irradiation dosage. The EST isozymes expression of M1 generation pollen was obviously changed by electron beam irradiation confirmed by use of PAGE electrophoresis. EST patterns of pollen of Gladiolus irradiated at the dose no more than 120Gy were similar to CK, but there were some differences in the vigor of isozymes. The EST bands added or absent had been observed at the dose more than 160Gy compared to CK. There were a special EST band more and 2 EST bands less in plants treated by both 160Gy and 200Gy electron beam irradiation, and a special EST band more and 1 EST bands less in plants treated by 240Gy electron beam irradiation. The vigor of isozymes of plants treated at the dose more than 160Gy showed some respective differences compared to the other treated and CK's. These results revealed that electron beam irradiation is an effective way for Gladiolus breeding.