Cholesterol biosynthesis is under tight control by sterol sensing proteins and transcriptional regulation; however, recent evidence suggests that this pathway may also be post-transcriptionally regulated by microRNAs (miRNA). miR-455-3p was found to be significantly up-regulated in mouse livers with high-fat diets, which coincided with significant repression of miR-455-3p target genes, as determined by cumulative distribution fraction analysis of gene expression microarray data. Ten of fourteen cholesterol biosynthetic enzymes were also found to be significantly down-regulated in the livers of these mice; however, sterol regulatory element binding transcription factor 2 (SREBF2) activity was found to be significantly increased, not decreased. Multiple enzymes in the cholesterol biosynthetic pathway were predicted to be miR-455-3p targets; therefore, we hypothesized that miR-455-3p is a master regulator of cholesterol production through direct and indirect targeting of enzymes in the biosynthetic pathway. Furthermore, inhibition of miR-455-3p with locked-nucleic acids (LNA) significantly increased mRNA levels of all cholesterol enzymes analyzed in vitro, including HMGCR, CYP51A1, DHCR7, SC4MOL, and SC5DL. Conversely, miR-455-3p over-expression resulted in decreased mRNA levels of specific cholesterol biosynthesis enzymes. Most importantly, miR-455-3p inhibition significantly increased cholesterol biosynthesis in multiple cell lines from multiple species, as determined by radiolabeled acetate incorporation assays; whereas miR-455-3p transfection decreased cholesterol biosynthesis. Using gene reporter (luciferase) assays, miR-455-3p was found to directly target the 3’ untranslated region of 7-dehydrocholesterol reductase (DHCR7). Inhibition of miR-455-3p in DHCR7 mutated cell lines representing Smith-Lemli Opitz Syndrome, which results from non-complete dysfunction of DHCR7, significantly increased DHCR7 mRNA levels and cholesterol biosynthesis. In summary, miR-455-3p is a powerful inhibitor of cholesterol biosynthesis through post-transcriptional regulation that likely overrides SREBF2 transcriptional regulation within cholesterol metabolism.