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Radiofrequency radiation, emitted from commonly used wireless communication devices, has been implicated in disrupting cellular homeostasis; however, its effects on testicular somatic cells such as Leydig cells remain poorly understood. To address this, the present study investigated the frequency- and time-specific effects of RFR on cellular morphology, proliferation, and cell cycle dynamics in TM3 Leydig cells. Cells were exposed to mobile phone radiation and radiofrequency signals at 1800 MHz and 2450 MHz for 15-120 min under non-thermal conditions. Following exposure, morphological alterations were examined using Giemsa staining, while proliferation and cell cycle progression were evaluated by BrdU-ELISA and PI-based flow cytometry. BrdU assays showed a progressive reduction in DNA synthesis across conditions, indicating suppressed proliferative activity. Consistently, cell cycle analysis revealed accumulation of cells in G1 phase with a corresponding decline in S-phase population at longer durations, suggesting checkpoint activation. These changes were supported by morphological alterations such as cell rounding, loss of adherence, and membrane blebbing, features associated with stress-induced antiproliferative responses. Overall, these findings indicate that RFR disrupts cellular morphology, DNA synthesis, and cell cycle progression in a frequency- and time-dependent manner, highlighting Leydig cell vulnerability to prolonged exposure and potential implications for male reproductive health.