Bacterial infections of the uterus or mammary gland commonly cause disease and infertility by perturbing growth and steroidogenesis of the dominant follicle in the ovary of cattle. Cells of the innate immune system use Toll-like receptors TLR2, TLR4, and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) expressed by bacteria, leading to activation of MAPK and nuclear factor-κBκ pathways and production of inflammatory cytokines such as IL-1β and IL-6, and the chemokine IL-8. The present study tested whether granulosa cells from dominant follicles have functional TLR2, TLR4, and TLR5 pathways. Supernatants of primary bovine granulosa cells accumulated IL-1β, IL-6, and IL-8 when treated for 24 hours with Pam3CSK4 (PAM) that binds TLR2 or lipopolysaccharide (LPS) that binds TLR4 but not flagellin that binds TLR5. Granulosa cell responses to PAM or LPS were rapid, with increased phosphorylation of p38 and ERK1/2 within 30 minutes and increased abundance of IL6, IL1B, IL10, TNF, IL8, and CCL5 mRNA after 3 hours of treatment. Accumulation of IL-6 in response to PAM and LPS was attenuated using small interfering RNA targeting TLR2 and TLR4, respectively. Furthermore, treating granulosa cells with inhibitors targeting MAPK or nuclear factor-κB reduced the accumulation of IL-6 in response to LPS or PAM. Treatment with LPS or PAM reduced the accumulation of estradiol and progesterone, and the PAMPs reduced granulosa cell expression of CYP19A1 mRNA and protein. In conclusion, bacterial PAMPs initiate inflammation and perturb the endocrine function of bovine granulosa cells from dominant follicles via TLR2 and TLR4 pathways.