Liver serine/threonine kinase B1 (LKB1) is a tumor suppressor associated with the pathogenesis of Peutz-Jeghers syndrome. Affected males are at increased risk of developing Sertoli cell tumors and display defective spermatogenesis. Male mice lacking the short isoform (Lkb1S) of Lkb1 were sterile and exhibited abnormal spermiogenesis. In addition to the short isoform, the long isoform of Lkb1 (Lkb1L) is also expressed in testis; however, the requirement of the long isoform for fertility and the functional difference between the isoforms remain unknown. Herein, different from the spermiation failure reported in Lkb1S knockout mice, conditional deletion (cKO) of both isoforms of Lkb1 in germ cells resulted in male sterility stemming from defects in acrosome formation, as well as nuclear elongation and condensation during spermatid differentiation. Additionally, cKO mice showed a progressive germ cell loss that was never reported in mice with Lkb1S deletion. Further experiments revealed that the defect resulted from the failure of spermatogonial stem/progenitor cells (SPCs) maintenance. Although increased mTORC1 activity in postnatal cKO testes was consistent with a tendency toward germline stem cell differentiation, in vivo inhibition of the pathway by rapamycin treatment failed to rescue the phenotype. Concurrently, we detected a significant reduction of mitochondrial activity in Lkb1deficient SPCs. The results suggest that the regulation of LKB1 on SPCs' maintenance is associated with mitochondrial functions but not through the mTOR signaling pathway. In summary, our study supports different roles of Lkb1 isoforms in spermatogenesis with Lkb1L directing SPCs maintenance, and Lkb1L and Lkb1S coordinately regulating spermatid differentiation.