Earlier studies have shown adjudin to cause aspermatogenesis by depleting virtually all germ cells from the seminiferous epithelium, leading to transient infertility; spermatogenesis and fertility were re-established several weeks later after germ cell proliferation and differentiation were reinitiated by spermatogonia. While adjudin is known to exert its initial effects at the apical ectoplasmic specialization (a testis-specific atypical anchoring junction), thereby perturbing spermatid adhesion, its molecular target(s) at this site is not known. Herein, we report the production of a specific antibody against adjudin after this compound was conjugated to an adjuvant (i.e., keyhole limpet hemocyanin) to maximize immuneresponse in rabbits. This antibody was utilized for co-immunoprecipitation by using an affinity resin to pull-down the binding partners of adjudin. Using this approach coupled with mass spectrometry and immunoblotting, we show testin (a protein largely restricted to the apical ES in the adult testis) and actin-myosin to be molecular targets of adjudin. These findings provide a platform for future functional studies, not only to better understand the molecular mechanism behind adjudin-induced germ cell loss from the seminiferous epithelium, but also to understand the molecular basis of spermiation.