Microglia, CNS resident innate immune cells, respond strongly to activation of TLR3 and TLR4, which recognize viral dsRNA poly(I:C) and bacterial endotoxin LPS, respectively. However, few studies have thoroughly and parallelly compared functional phenotypes and downstream mechanisms between LPS- and poly(I:C)-exposed primary microglia. Here, we investigated the responses of mouse primary microglia upon LPS and poly(I:C) stimulation by detecting various phenotypes ranging from morphology, proliferation, secretion, chemotaxis, to phagocytosis. Furthermore, we explored their sequential gene expression and the downstream signal cascades. Interestingly, we found that the microglial activation pattern induced by LPS was distinguished from that induced by poly(I:C). Regarding microglial morphology, LPS caused an ameboid-like shape while poly(I:C) induced a bushy shape. Microglial proliferation was also facilitated by LPS but not by poly(I:C). In addition, LPS and poly(I:C) modulated microglial chemotaxis and phagocytosis differently. Furthermore, genome-wide analysis provided gene-level support to these functional differences, which may be associated with NF-κb and type I interferon pathways. Last, LPS- and poly(I:C)-activated microglia mediated neurotoxicity in a co-culture system. This study extends our understanding of TLR roles in microglia and provides insights into selecting proper inflammatory microglial models, which may facilitate identification of new targets for therapeutic application.