Introduction: A growing body of evidence suggests that vitamin D status may be associated with clinically important outcomes in critically ill patients. However, the majority of this evidence is based on retrospective analyses of mixed (surgical and medical) ICU cohorts. Therefore, our goal was to: 1) prospectively characterize vitamin D status at initiation of critical care in surgical ICU patients; and 2) to determine whether this vitamin D status is associated with the risk of prolonged hospital (LOS), 90-day readmission, and/or 90-day mortality. Methods: We prospectively enrolled 100 patients from 2 surgical ICUs at a Boston teaching hospital. Baseline vitamin D assessment was performed within 24 hours of admission. We tracked the health status of enrolled subjects for a maximum of 90 days. Receiver operating characteristic (ROC) curves were generated to compare total 25-hydroxyvitamin D [25(OH)D], total 1,25-dihydroxyvitamin D [1,25(OH)2D], bioavailable 25(OH)D, and bioavailable 1,25(OH)2D as predictors of prolonged hospital LOS, 90-day readmission, and 90-day mortality. To test for the association of vitamin D status with prolonged hospital LOS, we performed a zero truncated Poisson regression analysis and adjusted for biologically plausible covariates. Similarly, to test for the association of vitamin D status with 90-day readmission and 90-day mortality, we performed multivariable logistic regression analyses and adjusted for biologically plausible covariates. Results: Mean (±standard deviation) serum total 25(OH)D and 1,25(OH)2D levels were 17 ± 8 ng/mL and 32 ± 19 pg/mL, respectively. Mean calculated bioavailable 25(OH)D and 1,25(OH)2D were 2.5 ± 2.0 ng/mL and 6.6 ± 5.3 pg/mL, respectively. ROC curve analysis demonstrated that all four vitamin D measures predicted the 3 clinical outcomes; total 25(OH)D was not inferior to the other measures. Median (interquartile range) hospital LOS was 11 (8–19) days. After adjusting for biologically plausible covariates, zero truncated Poisson regression analysis demonstrated an association of total 25(OH)D with hospital LOS [incident rate ratio per 1 ng/mL, 0.98 95% confidence interval (CI): 0.97–0.98]. 90-day readmission and 90-day mortality rates were 24% and 22%, respectively. After adjusting for biologically plausible covariates, there remained a significant association of total 25(OH)D with 90-day readmission (odds ratio (OR) per 1 ng/mL, 0.84; 95%CI: 0.74–0.95) and 90-day mortality (OR per 1 ng/mL, 0.84; 95%CI: 0.73–0.97). Conclusions: Total serum 25(OH)D levels within 24 hours of ICU admission may identify patients at high risk for prolonged hospital LOS, 90-day readmission, and 90-day mortality. Randomized trials are needed to assess whether vitamin D supplementation can improve these clinically relevant outcomes in surgical ICU patients.