Background: Although growing number of studies have explored the role of vitamin D₃ on various cancer types, no study has been conducted in the prospective cohort of advanced BTC. This analysis aimed to evaluate the predictive or prognostic implication of serum vitamin D₃ in pts with advanced BTC treated with second-line chemotherapy. Methods: The analysis is the preplanned subgroup analysis of the NIFTY trial, a multicenter, open-label, randomized, phase 2b study which compared the second-line liposomal irinotecan plus 5-FU/leucovorin and 5-FU/leucovorin in pts with advanced BTC. Serum vitamin D₃ (25-hydroxyvitamin D [25(OH)D]) levels were measured at baseline (i.e., C1D1) in a total of 176 pts out of 178 pts in the ITT population and were included in the current analysis. Correlation between OS and serum vitamin D₃ as a continuous variable was analyzed for linear and non-linear associations. Results: The median age was 64 years (range, 37–84) and 75 patients (42.6%) were women. The baseline serum vitamin D₃ levels ranged from 1.39 ng/ml to 140.02 ng/ml in the overall pts. There was no significant difference in the mean vitamin D₃ concentration according to the sex (male vs. female: 24.1 vs. 26.7; p=0.43), body mass index (BMI) (<25 vs. 25-29.99 vs. ≥30: 24.0 vs. 30.0 vs 15.9; p=0.18), previous surgery (no vs. yes: 24.1 vs. 27.4; p=0.40), ECOG performance status (0 vs. 1: 24.1 vs. 25.5; p=0.66), and primary tumor site (intrahepatic vs. extrahepatic vs. gallbladder: 25.7 vs. 23.2 vs. 26.2; p=0.74). After adjustment for key prognostic factors including chemotherapy regimens, metastatic sites, or baseline CA 19-9 levels, there was no significant association between baseline serum vitamin D₃ and OS (HR=1.06 per 10 increase; 95% CI, 0.97–1.15; p=0.22). In addition, there was no non-linear association between vitamin D₃ and OS (p=0.52 for the linear model and p=0.62 for the model with restricted cubic splines). In the subgroup analysis, higher vitamin D3 levels were associated with poorer OS (HR=1.16 per 10 increase; 95% CI, 1.04–1.30; p=0.0072) in female pts. Conclusions: Serum vitamin D₃ levels did not show significant relationship with survival in pts with advanced BTC treated with second-line chemotherapy. High vitamin D₃ levels might be associated with poorer survival in female pts. Further larger studies are needed.