Non-obstructive azoospermia is a severe condition because spermatogenesis per se is disrupted. Although microdissection testicular sperm extraction is the standard therapy for non-obstructive azoospermia, sperm retrieval is unsuccessful in approximately 50% of patients. For these patients, we conducted human chorionic gonadotropin-based salvage hormonal therapy, and sperm retrieval was possible in 10-20% of patients. The objectives of this study were to assess the changes in interstitial lesions in patients with nonobstructive azoospermia and to evaluate the effects of human chorionic gonadotropin on these tissues. Testicular biopsy specimens were obtained from 10 non-obstructive azoospermia patients who failed to obtain spermatozoa and from 10 obstructive azoospermia patients. All non-obstructive azoospermia patients received salvage hormonal therapy after microdissection testicular sperm extraction. Hematoxylin and eosin (H.E.) staining and immunohistochemical staining for steroidogenic acute regulatory protein antibody, the Leydig cell marker, and TE-7 antibody, the fibroblast marker, as well as picrosirius red staining to detect collagen fibers, were performed. We measured interstitial lesions, as Leydig cell area and the other area, with ImageJ software. Interstitial area, excluding Leydig cells, increased up to 12.5% in non-obstructive azoospermia compared with 1.2% in obstructive azoospermia (p < 0.01), which was mainly because of fibrosis with TE-7-positive fibroblasts. The increase in interstitial lesions was correlated with Johnsen scores. Interstitial area, excluding the Leydig cells, decreased by 29% after salvage hormonal therapy (p < 0.05), indicating improvement of interstitial fibrosis in non-obstructive azoospermia. There were no significant difference in total Leydig cell area and size of each Leydig cells between obstructive azoospermia and non-obstructive azoospermia. After the salvage hormonal therapy, a portion of the Leydig cells became hypertrophied and mean diameter of Leydig cell significantly increased (p < 0.01). This study showed anti-fibrotic effects of human chorionic gonadotropin and hypertrophic change of Leydig cells after human chorionic gonadotropin administration.