BACKGROUND: The osteoclastogenesis RANKL gene plays a key role in bone remodeling. The hypomethylation of its promoter region may cause osteoporosis. The present study aimed to elucidate the influence of physical activity on DNA methylation changes of RANKL promoter cytosine-phosphate-guanine (CpG)-rich region in active and sedentary adults and to assess the effect of aerobic and strength training on RANKL DNA methylation changes among Tunisian-North African adults. METHODS: A total of 104 participants including 52 adults (58\% males and 42\% females) and 52 adults (31\% males and 69\% females) were recruited for the observational and interventional part of the study, respectively. The intervention consisted of 12 weeks of aerobic training (30 min/session) followed by 10 minutes of strengthening exercises. All participants completed the International Physical Activity Questionnaire and provided blood samples for quantitative methylation-specific polymerase chain reaction (PCR) analysis. RESULTS: The study revealed a significant difference (P = 6 × 10-10) in the methylation level of the RANKL promoter region between active and sedentary adults, with a 6.68-fold increase observed in the active group. After the intervention, both the trained (P = 41 × 10-5) and untrained (P = .002) groups displayed high methylation levels in the RANKL promoter region. In addition, the trained group exhibited significant improvements in heart rate (P = 2.2 × 10-16), blood pressure (P = 39 × 10-3), maximal oxygen uptake (P = 1.5 × 10-7), and fat mass (P = 7 × 10-4). CONCLUSION: Exploring epigenetic modifications in the RANKL promoter region may contribute to a more comprehensive understanding of the complexity of osteoporosis. This suggests that aerobic/strength training could potentially improve the bone system, reducing its vulnerability to osteoporosis by increasing RANKL DNA methylation.