Background: Up to 40% of women with estrogen positive (ER+) breast cancer treated with aromatase inhibitors (AIs) experience musculoskeletal symptoms (AIMSS) such as bone pain, joint pain, joint stiffness, and muscle weakness that negatively impact functional ability. Previous reports indicate that vitamin D insufficiency is common in this patient population and that D supplementation might attenuate AIMSS by affecting muscle, bone and/or nerve metabolism. To test the association between AIMSS and serum 25(OH)D levels, we analyzed data collected as part of a pilot prospective study assessing instrument effect sizes to measure AIMSS. Methods: Data were analyzed from 58 women, age 61 ± 10 y with a history of ER+ breast cancer stage I-IIIa. AIMSS symptoms were measured at baseline (pre AI) and 6 mo using the Breast Cancer Prevention Trial Musculoskeletal Subscale (BCPT-MS), the Western Ontario and McMaster Universities Arthritis Index (WOMAC), and the Australian-Canadian Hand Osteoarthritis Index (AUSCAN). Serum 25(OH)D was measured by radioimmunoassay (Diasorin liaison, Stillwater, MN). Linear regression analysis was used to determine the association between changes in AIMSS scores (dependent variables) and changes in serum 25(OH)D from baseline to 6 mo, adjusting for age and season. Results: From baseline to 6 mo there were significant increases on all AIMSS measures (BCPT-MS, WOMAC and AUSCAN p<0.02). These increases in AIMSS measures were not associated with the change in serum 25(OH)D from baseline to 6 mo ( =11 ng/ml + 12; p<0.001). Conclusions: Worsening of symptoms during the initial 6 months of AI therapy was not associated with changes in serum 25(OH)D levels. These results suggest that short-term supplementation with 25(OH)D does not attenuate the symptoms of AIMSS in post-menopausal women with ER+ breast cancer during the first 6 mo of AI therapy. However vitamin D insufficiency was uncommon in this study population (mean 25(OH)D at baseline 31 ng/ml ± 10 and 42± 13 ng/ml at 6 mo). Well controlled clinical trials are needed to further our understanding of these relationships.