Pathogen-associated molecular patterns (PAMP) are highly conserved structural motifs that are recognized by pathogen recognition receptors (PRR) to initiate immune responses. Treatment of cells with double-stranded RNA (dsRNA) mimics viral infection and regulates expression of various genes. Polyinosinic-polycytidylic acid (Poly I:C) is a PAMP for viral infections. It is established that Poly I:C mediates the production of cytokines by binding to its PRR, toll-like receptor 3 (TLR3) on gamma-delta T cells. Because galectins are also involved in immune responses and the outcome of microbial infections, it was important to evaluate whether its production is affected by poly I:C stimulation in whole blood. The effect of Poly I:C, on the expression of galectins in cow blood was assessed in this study. Galectins are multipotent, evolutionarily conserved, carbohydrate-binding proteins that trigger a cascade of transmembrane signaling events such as cell activation, cytokine secretion, migration, and apoptosis. Blood was taken from 5 multiparous Holstein cows and incubated with 12.5 µg/mL of Poly I:C at 37°C, with 85% humidity and 5% CO₂ for 30 min. Total RNA was isolated, reverse-transcribed to cDNA, and then used in real-time PCR experiments with the use of commercially sequenced cow specific primers. GAPDH and β-actin served as internal controls. Fold change in transcript abundance was calculated using the Livak method. Where ΔCt = (Target genes _treat – GAPDH/β actin _treat) – ΔCt (Target genes _PBS – GAPDH/β actin _PBS). Fold change = 2^(-ΔΔCt). Concentrations of Galectins in plasma were measured using bovine Enzyme-linked Immunosorbent Assay(ELISA). Galectin concentration was analyzed with Proc GLM in SAS 9.4. Poly I:C increased the transcription of LGALS1, LGALS4, and LGALS8 (1.78,1.88, and 1.73 folds respectively). Secretion of Gal-1, -3, -8, and -9 were increased in treated samples compared with control (P = 0.0.10, P = 0.10, P < 0.0001, P < 0.0001, respectively). The results demonstrate that Poly I:C differentially modulates transcription of mRNA and secretion of galectins in cow blood. Elucidation of the relationship between PAMPS and galectin expression may help to define their roles in viral diseases as well as aid in drug design in the dairy industry.