A transcriptomic approach was used to determine the physiological mechanisms associated with the observed protective effect of a Saccharomyces cerevisiae fermentation product (NTK, NutriTek, Diamond V, Cedar Rapids, IA) during a mastitis challenge. Eighteen midlactation multiparous Holstein cows (n = 9/group) were fed a control diet (CON) or CON supplemented with 19 g/d NTK for 45 d, and then infected in the right rear quarter with 2500 cfu of S. uberis. Mammary gland tissue was collected from the infected quarter at 36 h via repeated percutaneous puncture, before antibiotic treatment was started to stop the infection. RNA was extracted and sequenced on the Illumina HiSeq 4000 system, generating an average of 25 million reads/sample, of which 95% were uniquely mapped, with 77% of gene-assigned read. A linear model with diet as fixed and animal as random effect was fitted. Differentially expressed genes (DEG) were declared at fold change ≥ |2| and P-value ≤0.05, identifying 150 DEG. The Dynamic Impact Approach was used for pathway analyses to determine effect on biological pathways. NTK supplementation activated genes related to immune cell recruitment (e.g., GRO1 and G-CSF), and increased innate immune cell antibacterial function (e.g., TNF, NOS2, CATHL4). Furthermore, NTK increased expression of genes involved in epithelial tissue protection (e.g., IL17C) and anti-inflammatory activity (e.g., HSP, BAG3, ATF3, p21). Pathway analysis indicated upregulation of tumor necrosis factor, heat shock protein response, and p21 pathways in the response to a mastitic event in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK fed cows at 36 h post challenge. Together with the lower quarter somatic cell count and temperature observed during the challenge (presented in a companion abstract), these data shed a light on the possible mechanism of action of NutriTek supplementation in increasing udder health during an intramammary challenge.