Abstract: This study investigated fertilization of spermatozoa of Misgurnus. anguillicaudatus at different time periods using optical microscope and electron microscope technology methods. The results showed that no acrosome was observed in the head of the sperm, which was mainly occupied by the nuclear. The implantation fossa of the sperms was low, and the mid-piece contained asymmetric sleeve. The flagella without lateral fins were organized as a 9+2 microtubular structure by axonemes. There was only one micropyle in shell of egg's animal pole, and the micropyle belongs to the type of concave and short channel. Under the water temperature of 20-21, sperm passed micropylar tube and entered ovum at 3s after insemination; sperm star light was observed at 5-8s; the second meiosis of the ovum occurred at 70s after insemination; the second polocyte appeared at 6-8min after insemination. Male and female prnucleus were fused at 20-25min after insemination. Zygote was in metaphase of first mitosis at 25-30min after insemination. The first mitosis finished at 40-45min after insemination, and two cells then formed. The inner diameter of micropylar tube of mature egg was (2.2860.364) m, similar like the diameter of sperm head, and the monospermy of M. anguillicaudatus. Eggs of M. anguillicaudatus were fertilizable in 150s after entering to water.