Vitamin D3 (VD3) is an essential immunomodulatory nutrientfor fish, but it is toxic at high doses. This study aimed to examine interactions between VD3 and bacterial lipopolysaccharide (LPS) endotoxin using larval zebrafish (Danio rerio). Larvae were pre-exposed to VD3 at 72 hours post fertilization (hpf) and at 96 hpf the media was replaced with LPS solutions; survival was assessed at 120 hpf. Total RNA was extracted at 72 and 96 hpf to quantify expression of VD3-metabolizing enzymes and transcriptomic changes using quantitative PCR and RNA-sequencing, respectively. Pre-exposure to 5, 10 and 20 µg/mL VD3 significantly reduced survival to LPS challenge in a dose-dependent manner. Basal expression of cyp2r1, cyp27a1, cyp27b1, cyp3a65, and cyp11a1 was upregulated between 72 and 96 hpf. VD3 exposure upregulated cyp24a1 expression and downregulated cyp2r1, cyp27b1, and cyp3a65 expression. Transcriptomic analysis revealed differentially expressed genes involved in inflammation, stress, and cell death positively correlated with endotoxin chemosensitization.