Background and Objectives: Vitamin D plays an important role in bone and muscle function, and cell prolifera-tion. The impact of chemotherapy and associated behavioural changes such as fatigue and sun avoidance on vit-amin D (25(OH) D) is unknown. This study aims to evaluate variations in serum vitamin D during chemotherapy and the predictive value of latitude, season and pre-existing vitamin D deficiency. Methods and Study Design: A 12-week prospective cohort study was conducted in chemotherapy-naïve patients in two Australian locations with different sun exposure. Vitamin D deficiency was defined as ≤ 25 nmol/L and insufficiency 26-50 nmol/L 25(OH) D. Demographics, chemotherapy regimen, nutritional status, sun exposure, geographic location, and sea-son were collected at baseline, 6 and 12 weeks after commencing chemotherapy. Results: Eighty-five patients (μ55.3±13.4 years of age; 49% female) were recruited, 96% Caucasian. Fifty-four patients were treated with cura-tive intent (mostly for breast [n=29] or colorectal [n=12] cancers). At baseline, 10 patients were vitamin D defi-cient and 33 were insufficient. Mean serum 25(OH) D (nmol/L) was higher at latitude -27.5o (Brisbane) than lati-tude -34.9o (Adelaide) (μ61.9±22.1 vs μ42.2±19.2, p < 0.001) and varied according to season (spring: μ46.9±20.3, summer: μ50.8±18.2, autumn: μ76.4±25.2, winter: μ36.5±15.7, p < 0.001). Serum 25(OH) D decreased with chemotherapy (baseline: μ49.2±22.3, 6-weeks: μ40.9±19.0, 12-weeks: μ45.9±19.7, p=0.05), with a significant and more rapid decline in winter and autumn (p=0.03). Conclusions: Chemotherapy is associated with a decrease in serum vitamin D, particularly during winter and autumn. Investigations into the underlying mechanism and as-sociated potential outcomes with this decrease requires further investigation.