The endoplasmic reticulum (ER) is a subcellular compartment where most secreted and membrane proteins are synthesized, folded, and post-translationally modified. Numerous pathologies, including diabetes and ischemic heart disease are associated with impaired ER protein-folding, which can lead to the accumulation of potentially toxic misfolded proteins (ER stress), and ultimately, cell death. The adaptive ER stress response, also known as the unfolded protein response (UPR), is an intracellular process that restores ER protein folding homeostasis. However, failure of the adaptive ER stress response to resolve ER stress leads to the maladaptive ER stress response, which guides the cell to programmed cell death. One of the proteins induced by the UPR during ER stress is MANF (mesencephalic astrocyte derived neurotrophic factor). MANF is a ubiquitously expressed, structurally unique, ER-resident protein. Secreted MANF protects cardiac myocytes from ischemic damage; however, the function of MANF within the ER is unknown. Accordingly, the objective of this study was to examine the function of MANF in the ER. The hypothesis was that, if intracellular MANF has a function in the ER, then decreasing its level in cultured cells would affect ER function. Accordingly, the approach of this study was to examine how siRNA-mediated decreases of MANF in cultured cells affect the adaptive and maladaptive ER stress responses. Activation of the ER stress response is mediated by three ER-transmembrane proteins, ATF6, PERK, and IRE-1, which sense ER protein misfolding and increase ER protein folding resources, such as the chaperones GRP94 and GRP78. IRE-1 activation that results from chemically induced ER stress was increased with MANF knockdown; however, ATF6 activation was unaffected by MANF knockdown. Surprisingly, MANF knockdown increased PERK activation, more so than chemically induced ER stress. Furthermore, upon chemically-induced ER stress, MANF knockdown increased GRP78 and GRP94. However, MANF knockdown did not affect cell viability, with or without chemically induced ER stress. Since reducing MANF increased the ER stress response, and since increases in ER stress occur when ER protein-misfolding occurs, then MANF must be required for optimal ER protein-folding.