Vitamin D status may be an important determinant of health-related quality of life of colorectal cancer (CRC) survivors. The current study investigated longitudinal associations between serum 25-hydroxyvitamin D3 (25OHD3) concentrations and quality of life in stage I-III CRC survivors up to 2 years post-treatment.CRC patients (n=261) were included upon diagnosis. Home visits (including blood sampling) were performed at diagnosis and at 6 weeks, 6 months, 1 and 2 years post-treatment. Serum 25OHD3 concentrations were measured using liquid chromatography-tandem mass spectrometry and adjusted for season. Validated questionnaires were used to assess global quality of life and cognitive functioning (EORTC-QLQ-C30), fatigue (EORTC-QLQ-C30 and Checklist Individual Strength, CIS), and depression and anxiety (Hospital Anxiety and Depression Scale). Statistical analyses were performed using linear mixed-models and adjusted for sex, age, time since diagnosis, therapy, comorbidities, physical activity, and BMI.At diagnosis, 45% of patients were vitamin D deficient (<50 nmol/L). After treatment, 25OHD3 concentrations increased on average with 3.1 nmol/L every 6 months. In confounder-adjusted models, 20 nmol/L increments in 25OHD3 were longitudinally associated with increased global quality of life (β 2.9; 95%CI 1.5,4.3) and reduced fatigue (EORTC-QLQ-C30 subscale: β -3.5; 95%CI -5.3,-1.8 and CIS: β -2.8; 95%CI -4.7,-0.9). Observed associations were present both within and between individuals over time.Higher concentrations of 25OHD3 were longitudinally associated with better global quality of life and less fatigue in CRC survivors.This study suggests that higher 25OHD3 concentrations may be beneficial for CRC survivors. Intervention studies are needed to corroborate these findings.