Neutrophils are cells of the innate immune system that have the ability to respond to stimuli. Galectin-8, a part of the family of galectins, has been shown to modulate the innate and adaptive immune system. The objective of this study was to analyze the effect of exogenous galectin-8 on global transcription in bovine neutrophils. Whole blood was collected from the jugular vein of clinically healthy Holstein-Friesian cows from the North Carolina A&T State University Dairy Unit (n = 5). Neutrophils were isolated by differential centrifugation and hypotonic lysis and TC20 was used to measure viability. Neutrophils were then treated (1 × 10⁶ cells/mL viable) with rGal-8 (2 μg), LPS (10 μg), rGal-8 + LPS, or maintained in PBS at 37°C for 1 h, 5% CO₂. Total RNA was extracted using Trizol, RNA integrity (RIN) was determined using Agilent Bioanalyzer, and RNA with RIN of >7 was used. The RNA sequences were generated using an Illumina HiSeq 4000 sequencer. Short RNA fragment reads were in FASTQ format and aligned to the cow reference genome (bosTau8), using Spliced Transcripts Alignment to a Reference (STAR) software. Mapped reads were counted with HTSeq. Genes were normalized against the control, PBS. Differentially expressed genes were analyzed with DESeq2. Pathway analysis was conducted using Ingenuity Pathway Analysis (IPA) software and Database for Annotation, Visualization and Integrated Discovery (DAVID). Our results show that 14,023 genes were expressed. Transcriptome profiling identified differentially expressed transcripts with the treatment of galectin-8 (2037), LPS (477), and Gal-8+LPS (1065) (P < 0.05). Galectin-8 targeted 78 pathways, including MAPK signaling pathway; Gal8+LPS targeted 63 pathways including TNF signaling pathway; and LPS targeted 41 pathways including toll-like receptor signaling pathway using DAVID. Integrin Signaling pathway was one of the top canonical pathways targeted by galectin-8 using IPA. These results suggest galectin-8 has broad interactions with several molecules modulating cytokines, chemokines, and innate and adaptive immune genes.