In recent years, declining male fertility and increasing incidence of testis-related diseases have become increasingly prominent, with environmental toxins, genetic factors, and lifestyle being the main contributing factors. Assessment of male reproductive toxicity has emerged as a critical link in environmental safety and drug development processes. Traditional animal experiments suffer from several limitations, including long experimental cycles, ethical controversies, interspecies differences, and the inability to simulate the testicular microenvironment.With the implementation of the 3R principle (reduction, replacement, and refinement), the construction of in vitro models with high physiological relevance has becomeinevitable. 3D cell culture can mimic the in vivo environment and provide a suitable 3D niche for testicular cells. This review summarizes the research progress into 3D cell culture for the development of testicular models. The composition of the testicular germ cell niche and its significance for toxicity assessment are detailed, and the characteristics and efficacy of five 3D culture systems including decellularized testicular scaffolds and hydrogel scaffolds, from the perspectives of their technical principles and applications are analyzed. Additionally, existing limitations of current models are addressed, proposing that future research should focus on standardization, multicell co-culture, and high-throughput screening. These efforts are intended to promote the application of 3D testicular models in reproductive toxicity research, providing support for environmental safety assessment and drug development.