Spermatozoa used for intracytoplasmic sperm injection (ICSI) are selected based on their motility and morphology. To explore other methods for selecting better spermatozoa, we developed a spermatozoa-sorting method using a physiologically natural selection system involving penetration into cervical mucus (CM). In addition, we analyzed the spermatozoa that penetrated the CM (CM-penetrating spermatozoa). The results were as follows. The CM-penetrating spermatozoa traveled a longer distance with better linear motility than spermatozoa in semen. Also, in comparison with spermatozoa obtained by density gradient centrifugation (DGC) and the swim-up method, the proportion of spermatozoa with normal morphology was higher, although, the proportion of spermatozoa with the head vacuole did not change. No DNA fragmentation was detected in the CM-penetrating spermatozoa. This method has several advantages. The technical procedure is simple and easy. Physical damage to spermatozoa is reduced because it does not require any centrifugation or washing procedure. A higher collection ratio of morphologically normal spermatozoon is achievable compared to the DGC and swim-up method, and it is a physiological selection method. We conclude that the CM penetration-based spermatozoa-sorting method is a promising new technique for ICSI because it is possible to collect physiologically better spermatozoa than those selected in the conventional selection method.