Accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress, which triggers the unfolded protein response (UPR) to restore protein homeostasis. So far, three UPR sensors have been identified, including IRE1, PERK, and ATF6. All of them are ER transmembrane proteins, which become activated and initiate downstream UPR signals under ER stress. Though first discovered during the study of how cells respond to ER stress, it remains unclear how the ER stress signal is sensed by the three UPR sensors. Structural studies provide insight into how direct binding of ER-localized peptides to the lumenal domain of IRE1 and PERK facilitates their oligomerization and thus activation. In another model, dissociation of the ER chaperone BiP is the key to the UPR activation. In addition, further studies reveal that the UPR not only functions in maintaining protein homeostasis and UPR sersors is not solely activated in response to the accumulation of unfolded proteins in the ER. Lipid bilayer stress, cytosolic factors or intercellular signals may initiate the UPR. Despite the importance of the UPR in physiology and pathophysiology, how the UPR is activated under physiological or pathophysiological conditions are largely unknown. Developing novel strategy to monitor the unfolded and misfolded proteins in the ER lumen will advance our understanding of the relationship between ER stress and the UPR. This paper introduces the discovery and the canonical pathways of the UPR, with a focus on the current mechanistic understanding of the UPR activation, and discusses the relationship between the UPR and ER stress as well as related questions.