Male reproductive development starts early during embryogenesis with the formation of spermatogonial stem cells (SSCs) from their neonatal precursors, the gonocytes. Previously, we reported high levels of CNNM1 expression in the testicular germ cells of neonatal mice, which were maintained in the SSCs of pubertal and adult mice. CNNM1 exhibited a progressive decline in expression levels as the SSCs progressed through the spermatogenic differentiation sequence. However, the specific functional role of CNNM1 during spermatogenesis remains elusive. Here, we studied the expression pattern of Cnnm1 during gonocyte to spermatogonia transition (GST), and expression levels were found to be higher at the initial establishment phase of SSCs marked with a higher rate of cell proliferation. GDNF-mediated self-renewal/proliferation of the C18-4 spermatogonial cell line enhanced the expression of CNNM1. Overexpression of CNNM1 in C18-4 spermatogonial cell lines resulted in the upregulation of genes involved in cell proliferation, nucleic acid metabolism, male germ cell development and various pathways involved in cell cycle regulation, whereas CNNM1 knockdown altered the cell cycle progression. Based on the expression analysis and proteome profiling, we conclude that CNNM1 regulates and maintains the self-renewal, proliferation, and survival of mouse spermatogonial cells.