The purpose of this study was to define the histopathological change and immunological response occurring as a result of testicular fine needle aspirations (TEFNAs) in C3H/HeN mice. TEFNA (one-, five-, or twenty-time aspiration) from the left testis was performed on day 0, and bilateral testes were examined histologically on days 21 and 49. Delayed-type hypersensitivity (DTH) was measured as the immunological response. Significant reduction of left testicular weight was observed only in the twenty-time aspiration group, on days 21 and 49. Histological examinations in the left testes showed a significantly high incidence of spermatogenetic disturbance with focal chronic inflammation and degenerative changes on day 21 and 49 after twenty-time TEFNA. Similar but less extensive changes were noted after five-time TEFNA. As for the weight and histology of the right testes, there was no significant change after the left-side TEFNA treatments. However, we revealed that the DTH response to autologous testicular cells was significantly elevated in the twenty-time aspiration group on day 21. In this animal model, TEFNA caused definite damage to the seminiferous tubules and induced DTH against testicular cells. Further study may be required to determine the clinical relevance of such findings to the testes of azoospermic men treated with TEFNA.