The testes are a sensitive organ to electromagnetic pollution and people are concerned about the harmful effects of the radiofrequency radiation (RFR) emitted from cellular phones. Therefore, the purpose of this study was to investigate the effects of long-term exposure to different RFR frequencies on single-strand DNA breaks and oxidative changes in rat testicular tissue. Twenty-eight male Sprague-Dawley rats were divided randomly into four groups. Three groups were exposed to radiation emitted from 900, 1800 and 2100 MHz RF generators, 2 h/day for 6 months. The sham-control group was kept under the same experimental conditions but the RFR generator was turned off. Immediately after the last exposure, testes were removed and DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) were analyzed. The results of this study indicated that RFR increased TOS, OSI, MDA and 8-OHdG (p < 0.05). TAS levels in the exposed group were lower than in the sham group (p < 0.05). In terms of DNA damage, the tail intensities in the comet assay were higher in the exposure groups (p < 0.05). This study demonstrated that long-term exposure to RFR emitted by cellular phones may cause oxidative stress and oxidative DNA damage in rat testicular tissue and may generate DNA single-strand breaks at high frequencies (1800 and 2100 MHz). Our results showed that some RFR emitted from cellular phones has potential to lead to cell damage in the testes.