Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long‐term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long‐term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum‐free culture conditions in the presence of glial‐cell‐line‐derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)‐like cell morphology, express pluripotent‐stem‐cell‐specific and germ‐cell‐specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low‐serum media in the presence of 6‐bromoindirubin‐3′‐oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ‐cell‐specific markers and pluripotent stem cell markers. These findings could be useful for the development of long‐term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.