As one of common characters in teleostean fish fertilization, spermatozoa pass through a tiny chorionic cavity named “micropyle” to reach their eggs. Micropyles appear to be a part of the barrier system for keeping species strictly. Additionally, in some sea water fishes, sperm attractants for same species are identified from micropyler region. However, it is not easy to observe sperm entry into its micropyle in many freshwater fish species, because spermatozoa usually cease moving within a few seconds in freshwater, and the egg size is usually over 5 mm in diameter and too large to find and observe a micropyle. To probe into this, we developed a system where spermatozoa with longevity and a part of chorionic micropyle were employed. The system allowed us to examine in detail how spermatozoa enter into micropyle in freshwater fishes. The vestibulum is a structure as a large funnels attached to the micropyle and is observed in many freshwater fish eggs. This structure seems lead spermatozoa to eggs. In salmonid fishes, even spermatozoa of different species swam around, traced on the surface of an egg, and were eventually guided into the micropyle. Another freshwater fish species, Tribolodon, also showed same phenomena. These results suggest that a micropyle does not keep species barrier, but rather effectively mobilize spermatozoa sparse in freshwater even different species. We discuss the role of micropyle in freshwater fish fertilization.