Updated project metadata. The mammalian Endoplasmic Reticulum Membrane protein Complex (mEMC) is an evolutionarily conserved hetero-oligomer whose function and mechanism of action remain elusive. Here we identify a multi-layered contribution of the mEMC to post-translational regulation of cholesterol homeostasis. Mammalian cells employ elaborate sensing and feedback mechanisms to balance free and stored cholesterol through biosynthesis, uptake, efflux and storage. We show that mEMC biogenesis is a prerequisite for optimal maturation of at least two key ER-resident sterol regulatory enzymes: squalene synthase (SQS) and sterol-O-acyltransferase (SOAT1). Without sufficient SQS, mEMC-deficient cells have attenuated cholesterol biosynthetic capability, and loss of SOAT1 significantly reduces the amount of stored cholesteryl esters (CEs). mEMC-deficient cells remain viable, maintaining free cholesterol levels with minimal transcriptional activation of cholesterogenic and uptake pathways by the sterol regulatory element binding protein (SREBP)-2. Consequently, cells lacking the mEMC were susceptible to changes in extracellular cholesterol availability and effectively were transformed into cholesterol auxotrophs. Supplementing mEMC-deficient cells with cholesterol or lanosterol failed to restore SQS or SOAT1. These data are consistent with a direct role for the mEMC in the biogenesis of SQS and SOAT1. The range of phenotypes observed previously with mEMC subunit depletion may result from its impact on fundamental cholesterol homeostatic mechanisms.