To investigate the effects of 5G mobile phone radiofrequency radiation (RFR) on sperm quality and sex hormone level of adult male mice, 24 healthy adult male C57BL/6 mice were randomly divided into two groups, the sham-exposure group (Sham) and 4.9 GHz-exposure group (4.9 GHz RFR), with 12 mice in each group. The mice in the exposure group were exposed to RFR with a power density of 50 W/m² and frequency of 4.9 GHz for 21 consecutive days, for 1 h a day. After exposure, the sperm quality was evaluated by detecting the sperm quantity and abnormality rate; Histomorphology of the testis was determined using Hematoxylin Eosin (HE) staining; the levels of testosterone (T), follicle stimulating hormone (FSH), luteinizing hormone (LH), gonadotropin releasing hormone (GnRH), glial derived neurotrophic factor (GDNF), and stem cell factor (SCF) were measured using enzyme-linked immunosorbent assay (ELISA); A western blot was used to detect the levels of GDNF, SCF and tight junction related proteins (ZO-1 and Occludin) of the blood-testis barrier (BTB) in testis tissue. Compared with the Sham group, no significant change in testicular morphology, sperm count, and sperm abnormality rate was observed in the 4.9 GHz RFR group; The content of FSH and LH in serum showed no significant differences; and the levels of GDNF, SCF, ZO-1 and Occludin in the testis showed no significant differences. However, the concentration of T and GnRH in serum decreased significantly. The results showed that under the experimental conditions, 4.9 GHz RFR had no significant effect on the sperm quality of mice, but could lead to changes in sex hormone levels.