A plethora of evidence supports the role of cyclic nucleotides in junction restructuring. For instance, studies have shown cGMP to be a key regulator of junction assembly and disassembly in different in vitro and in vivo systems. In this study, we examine the role of soluble guanylate cyclase (sGC) in junction restructuring in the seminiferous epithelium of the rat testis. First, the interaction of soluble guanylate cyclase β1 (sGCβ1; sGC is a heterodimer comprised of an α and a β subunit) with proteins that constitute adherens and tight junctions in the testis was demonstrated. By immunoprecipitation, sGCβ1 was found to associate with occludin, JAM-A, and ZO-1, as well as with cadherin, catenin, nectin, afadin, ponsin, and espin, suggestive of its role in cell junction dynamics. These results were corroborated in part by immunohistochemistry experiments, which revealed that the localization of sGCβ1 was largely restricted to the site of the apical and basal ectoplasmic specialization. Next, the role of sGC in junction dynamics was addressed by using an in vivo model of junction restructuring. Administration of Adjudin—a chemical entity known to specifically perturb adhesion between Sertoli and germ cells (i.e., round and elongate(ing) spermatids and most spermatocytes)—resulted in a ~1.5-fold increase in sGCβ1, coinciding with the loss of germ cells from the epithelium. More importantly, the ability of sGCβ1 to associate with cadherin increased approximately three-fold during Adjudin-mediated restructuring of Sertoli-germ cell junctions, whereas its interaction with tight junction proteins (i.e., occludin and ZO-1) decreased. Taken collectively, these results suggest that sGC participates in the remodeling of cell junctions during spermatogenesis.