Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial (NHBE) cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in NHBE cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knock-down, in a concentration-dependent manner. We further demonstrated that the anti-viral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour pre-incubation with the LSS inhibitor. The rank order of anti-viral potency of the LSS inhibitors used was consistent with LSS inhibition potency, however all compounds showed remarkably higher potency against RV compared to the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate anti-viral defense protein, interferon-β. We found LSS to be a novel regulator of RV replication and innate anti-viral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.