Epidemiological studies on the prevalence of musculoskeletal pain have consistently shown that this is a relevant health problem, with non-specific low back pain (LBP) being the most commonly reported in adult females. Conflicting data on the association between LBP symptoms and physical activity (PA) have been reported. Here, we investigated the prevalence of LBP and the effect of a 24-month non-specific PA intervention on changes in LBP prevalence in a series of Italian healthy postmenopausal women. We performed a secondary analysis in the frame of the DAMA trial, a factorial randomized intervention trial aimed to evaluate the ability of a 24-month intervention, based on moderate-intensity PA, and/or dietary modification, in reducing mammographic breast density in healthy postmenopausal women. The PA intervention included at least 1 hour/day of moderate PA and a more strenuous weekly activity, collective walks and theoretical group sessions. A self-administered pain questionnaire was administered at baseline and at the end of the intervention. The questionnaire was specifically structured to investigate the occurrence of musculoskeletal pain, the body localization, intensity and duration of the pain. Two hundred and ten women (102 randomized to PA intervention, 108 not receiving the PA intervention) filled out the questionnaires. At baseline LBP was present in 32.9% of the participants. Among women randomized to the PA intervention, LBP prevalence at follow up (21.6%) was lower than at baseline (33.3%) (p = 0.02), while in women who did not receive the PA intervention the LBP prevalence at baseline and follow up were 32.4% and 25.9%, respectively (p = 0.30). Overall, there was no significant between-group effect of PA intervention on LBP. Further studies are needed to understand the role of non-specific PA intervention, aimed to improve overall fitness, on LBP prevalence.