Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.