Muller cells, the principal glia of the retina, play several key roles in normal and various retinal diseases. To date, their direct involvement in retinal innate defense against bacterial pathogens has not been investigated. In this article, we show that Muller cells express TLR2, a key sensor implicated in recognizing Gram-positive bacteria. We found that intravitreal injection of TLR2 agonist Pam3Cys and Staphylococcus aureus activated Muller glia in C57BL/6 mouse retina. Similarly, Pam3Cys or S. aureus elicited the expression of TLR2 and activated the NF-κB and p38 MAPK signaling cascade. Concomitant with the activation of signaling pathways, transcriptional expression and secretion of various proinflammatory cytokines (IL-6, TNF-α, and IL-1β), chemokines (IL-8), and antimicrobial peptide (LL-37) were also induced in Muller glia. Importantly, the culture media derived from TLR2-activated Muller glia exhibited robust bactericidal activity against S. aureus. Furthermore, use of neutralizing Ab, small interfering RNA, and pharmacological inhibitors revealed that Muller glial innate response to S. aureus is mediated via the TLR2-NF-κB axis. Collectively, this study for the first time, to our knowledge, establishes that the retinal Muller glia senses pathogens via TLR2 and contributes directly to retinal innate defense via production of inflammatory mediators and antimicrobial peptides.