Citrobacter koseri (C. koseri) is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multi-focal brain abscesses. The roles of Toll-like receptor 4 (TLR4) and its signaling adaptor MyD88 during CNS C. koseri infection have not yet been examined, which is important since recent evidence indicates that innate immune responses are tailored towards specific pathogen classes. Here TLR4 WT (C3H/FeJ) and TLR4 mutant (C3H/HeJ) mice as well as MyD88 KO animals were infected intracerebrally with live C. koseri, resulting in meningitis and ventriculitis with accompanying brain abscess formation. MyD88 KO mice were exquisitely sensitive to C. koseri, demonstrating enhanced mortality rates and significantly elevated bacterial burdens compared to WT animals. Interestingly, although early proinflammatory mediator release (i.e. 12 h) was MyD88-dependent, a role for MyD88-independent signaling was evident at 24 h, revealing a compensatory response to CNS C. koseri infection. In contrast, TLR4 did not significantly impact bacterial burdens or proinflammatory mediator production in response to C. koseri. Similar findings were obtained with primary astrocytes, where MyD88-dependent pathways were essential for chemokine release in response to intact C. koseri, whereas TLR4 was dispensable; implicating the involvement of alternative TLRs since highly enriched astrocytes did not produce IL-1 upon bacterial exposure, which also signals via MyD88. Collectively, these findings demonstrate the importance of MyD88-dependent mechanisms in eliciting maximal proinflammatory responses, astrocyte activation, and bacterial containment during CNS C. koseri infection, as well as a late-phase MyD88-independent signaling pathway for cytokine/chemokine production.