Background: Vitamin D3 supplementation (D3) has been suggested as a treatment for aromatase inhibitor (AI)-associated musculoskeletal symptoms (AIMSS), but efficacy and safety are unclear. Methods: We randomly assigned 113 post-menopausal women ( 18 years; stage I-IIIA breast cancer; taking an AI and experiencing AIMSS), to either 600 IU D3 (control: n = 56) or 4,000 IU D3 (experimental: n = 57), daily for 6 months (6 mos). The primary study endpoint was change in musculoskeletal symptoms (MS) from baseline to 6 mos, measured by: The Breast Cancer Prevention Trial Symptom Scales-MS subscale (BCPT-MS), the Australian/Canadian Osteoarthritis Hand Index (AUSCAN), the Western Ontario and McMaster Osteoarthritis Index (WOMAC) and hand grip strength (Dynamometer). Plasma AI pharmacokinetics (AI-PK) were estimated using non-linear mixed-effects modeling. Effects of D3 on AI-PK were tested by likelihood ratio test. Serum 25(OH)D was quantified by chemiluminescent immunoassay (DiaSorin, Stillwater, MN). Sample size was calculated on a changeof 0.62 in BCPT-MS score. Assuming a two-tailed test with a = 0.05 and power = 80%, adequate sample size was 116 (58 per group). Primary endpoint analyses were based on intent-to-treat and determined using a General Linear Model controlling for possible effect modifiers. Results: The groups did not differ on demographic or clinical characteristics nor on AIMSS measures. After 6 mos, serum 25(OH)D was 33±8 ng/mL vs. 46±11 (mean±sd; control vs experimental; p < 0.001). There were no statistically significant differences between groups (control vs exp) in mean change in AIMSS scales from baseline to 6 mos: BCPT-MS: -0.45 vs. -0.24; WOMAC function: -1.23 vs -3.96; WOMAC pain: -0.56 vs. -1.18; WOMAC stiffness: -0.47 vs -0.54; AUSCAN function: -0.75 vs -1.12; AUSCAN pain: -0.24 vs. -0.90; AUSCAN stiffness: -0.11 vs. -0.08; hand grip: 1.06 vs 1.78 (all p > 0.1). AI clearance did not differ significantly between groups (baseline vs 6 mos; p > 0.5). Conclusions: Women randomly assigned to higher dose D3 (4,000 IU) showed no improvement in AIMSS over usual dose D3 (600 IU). While D3 does not appear to adversely affect AI drug metabolism, it may have other health effects in this population.