Acetaminophen is a non-narcotic analgesic used as an analgesic and antipyretic. Acetaminophen is used for mild to moderate pain; its efficacy is low as analgesic as compared to non-steroidal anti-inflammatory drugs (NSAIDs) as it has no any anti-inflammatory effect. Despite of its well-known use and safely, however; the precise mechanism of acetaminophen still enigmatic. Findings from preclinical studies suggest that the main mechanism of acetaminophen is related to the inhibition of cyclooxygenase 3 (COX-3) which is variant of COX-1 expressed in the brain. However, the profound analgesic antinociceptive effects of acetaminophen cannot depend merely on this pathway. Further findings from preclinical and clinical studies confirmed that acetaminophen and its metabolites can modulate different signaling pain pathways other than COX pathway. Thus, this review revises the potential mechanistic pathways of acetaminophen in relation to its clinical applications.