When stimulated with pathogenic and inflammatory signals, such as bacterial lipopolysaccharide (LPS), macrophages produce biologically active 1,25-dihyroxyvitamin D (1,25D) from circulating 25-hydroxyvitamin D. Among other signals, 1,25D signaling in macrophages strongly induces the expression of neutrophil chemoattractants, such as IL-8/CXCL8. In addition, our recent re-analysis of 94 human and mouse vitamin D-regulated expression profiles also suggested that 1,25D may regulate granule formation and chemotaxis in undifferentiated human promyelocytic leukemia HL60 cells. We were therefore interested in determining the effects of 1,25D alone and in combination with an inflammatory signal such as LPS on gene expression and function of primary human neutrophils. Preliminary studies indicated that primary neutrophils were 1,25D responsive (induction of CYP24A1, CAMP, CD14). LPS superinduced expression of CAMP, but strongly suppressed that of CYP24A1 in the presence of 1,25D, suggesting 1,25D catabolism is inhibited in the presence of LPS. We performed bulk RNAseq analysis of triplicate isolates of primary human neutrophils treated with 1,25D alone or in combination with LPS for 6h. Principal component analysis confirmed that each treatment condition produced distinct expression profiles and that those of each of the triplicates were highly concordant. LPS- and 1,25D-regulated gene expression profiles were largely distinct. ~900 genes were significantly up- or downregulated by 1,25D at least 1.5-fold, and a further 900 genes were regulated by 1,25D in combination with LPS compared to LPS or 1,25D alone. Pathways analyses, as well as Gene Ontology over-representation analysis for biological processes and cellular components, revealed a role for 1,25D (both on its own and in the presence of LPS) in diverse neutrophil innate immune functions, including degranulation, cell-cell adhesion and migration, and production of cytokines. Consistent with pathways analyses, mRNA expression of key genes involved in degranulation and migration are stimulated by 1,25D. Collectively, the data suggest that exposure to 1,25D enhances neutrophil bacterial killing. Our data also sheds new light on additional mechanisms by which vitamin D signaling regulates innate immune responses to infection.