Animals and plants are exposed to myriads of potential microbial invaders. In case of animals, Toll-like receptors (TLRs) act as the primary defense against infection by pathogens. Arguably, less is known regarding the activation of TLRs that connect the innate and adaptive immune systems. Some TLR ligands have been used as adjuvants in various vaccines and have gained a great deal of attention due to their ability to elicit an effective immune response. Understanding the intricate relationships between various molecules involved in TLR signaling and their positive or negative regulation is a key focus for the development of effective therapeutics. In this review, recent developments in TLR signaling that will be very important in providing new drug target molecules and a better understanding of molecular regulation of innate immunity are discussed.