Abstract: Extracellular membrane proteins play key roles in the interaction of testicular microenvironment germline stem cell (GSC) through mediating cell attachment, recognition, and signal transduction processes. Cadherin 18 (CDH18), a type II classical cadherin, is mainly expressed in the nervous and reproductive systems. Here we report the expression of CDH18 in the Prospermatogonia (ProSGs) of neonatal pigs and mouse spermatogonial stem cells (SSCs). Disruption of CDH18 expression showed no detrimental impact on cell morphology, proliferation, self-renewal or differentiation in cultured porcine ProSGs, but enhanced cell adhesion and prolonged maintenance. Transcriptomic analysis indicated that the downregulation of CDH18 in ProSGs significantly upregulated genes and signaling pathways associated with cell adhesion. To further reveal the function of CDH18 in germ cells, Cdh18 knockout mice were constructed, which exhibited normal testicular morphology, histology, and spermatogenesis. Moreover, transcriptomic data showed increased expression of genes associated with adhesion, consistent with those observed in porcine ProSGs. The interaction of CDH18 with β-catenin and JAK2 in both porcine ProSGs and murine SSCs indicated an inhibitory effect on the canonical Wnt and JAK-STAT signaling pathways during CDH18 deficiency. Collectively, our findings highlight the crucial role of CDH18 in regulating the cell adhesion in porcine ProSGs and mouse SSCs. These insights may have significant implications for understanding the regulatory mechanism of testicular niche.