Abstract Background: Vitamin D status may be an important determinant of health-related quality of life of colorectal cancer survivors. The current study investigated longitudinal associations between serum 25-hydroxyvitamin D3 (25OHD3) concentrations and quality of life in stage I–III colorectal cancer survivors up to 2 years after treatment. Methods: Patients with colorectal cancer (n = 261) were included upon diagnosis. Home visits (including blood sampling) were performed at diagnosis and at 6 weeks, 6 months, 1 year, and 2 years after treatment. Serum 25OHD3 concentrations were measured using LC/MS-MS 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 body mass index. Results: 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% confidence interval (CI), 1.5–4.3] and reduced fatigue (EORTC-QLQ-C30 subscale: β −3.5; 95% CI, −5.3 to −1.8 and CIS: β −2.8; 95% CI, −4.7 to −0.9). Observed associations were present both within and between individuals over time. Conclusions: Higher concentrations of 25OHD3 were longitudinally associated with better global quality of life and less fatigue in colorectal cancer survivors. Impact: This study suggests that higher 25OHD3 concentrations may be beneficial for colorectal cancer survivors. Future intervention studies are needed to corroborate these findings.