The overpopulation of abandoned and stray companion animals has become a global crisis. The main purpose of this study was to develop a novel nanomedicine-based antifertility compound for non-surgical castration of male animals. Mangosteen (Garcinia mangostana L) pericarp extract has been shown to exhibit anti-fertility property. α-mangostin (AM)-loaded nanostructured lipid carrier (AM-NLC) was developed to improve male germ cell apoptosis. This study was conducted to investigate physicochemical properties of AM-NLC and determine the biological effects of AM-NLC on spermatogonia cells and testicular explants obtained from castrated testes. AM-NLC was produced through a hot homogenization technique. The negatively charged particle of AM-NLC was nano-sized with a narrow dispersity. AM-NLC exhibited antiproliferative activity towards spermatogonium cells. It induced apoptosis in the cells. In addition, AM-NLC exhibited anti-inflammatory activities in lipopolysaccharide-activated macrophages. Abnormal anatomy of seminiferous tubule was noted following treatment of testicular explant with AM-NLC. This nanomedicine-based sterilant would be a promising platform that may have utility in non-surgical castration of male animals by intra-testicular injection.