ImportanceDaily supplementation with the probiotic Limosilactobacillus reuteri ATCC PTA 6475 (L reuteri) vs placebo has previously been demonstrated to reduce bone loss in an estrogen deficiency mice model and older women, although the magnitude of the effect was small. We hypothesized that long-term treatment with L reuteri could result in clinically relevant skeletal benefits in postmenopausal osteoporosis.ObjectiveTo evaluate whether daily supplementation with L reuteri vs placebo could reduce early postmenopausal bone loss and whether the effects remained or increased over time during 2 years of treatment.Design, Setting, and ParticipantsA double-blind, randomized, placebo-controlled clinical trial was conducted between December 4, 2019, and October 6, 2022, at a single center in Gothenburg, southwestern Sweden. Participants were recruited by online advertisements, and letters were sent to 10 062 women aged 50 to 60 years. Responding women (n = 752) underwent telephone screening, resulting in 292 women being invited to a screening visit. Of those who were screened, 239 women met all inclusion criteria and had no exclusion criteria.InterventionsCapsules with L reuteri in 2 doses, 5 × 108 (low dose) or 5 × 109 (high dose) colony-forming units, taken twice daily or placebo were administered. All capsules also included cholecalciferol, 200 IU.Main Outcomes and MeasuresThe primary outcome was the relative change in tibia total volumetric bone mineral density (vBMD) over 2 years. Secondary outcomes included relative change in areal BMD of the lumbar spine and total hip, bone turnover markers C-terminal telopeptide cross-links of collagen type I and type I procollagen intact N-terminal propeptide, as well as tibia trabecular bone volume fraction and cortical vBMD. Both intention-to-treat and per-protocol analyses were conducted.ResultsA total of 239 postmenopausal women (median age, 55 [IQR, 53-56] years) were included. Tibia vBMD (primary outcome), hip and spine vBMD, and tibia cortical area and BMD decreased significantly in all groups, with no group-to-group differences (percent change tibia vBMD high dose vs placebo least-squares means, −0.08 [95 CI, −0.85 to 0.69] and low dose vs placebo least-squares means, −0.22 [95% CI, −0.99 to 0.55]). There were no significant treatment effects on any other predefined outcomes. A prespecified sensitivity analysis found a significant interaction between body mass index (BMI) and treatment effect at 2 years. No significant adverse effects were observed.Conclusions and RelevanceIn this randomized clinical trial of 239 early postmenopausal women, supplementation with L reuteri had no effect on bone loss or bone turnover over 2 years. The observed interaction between BMI and treatment effect warrants further investigation.Trial RegistrationClinicalTrials.gov Identifier: NCT04169789