Hyperlipidaemia, i.e. increase in total cholesterol and triglycerides, is a common side‐effect of the immunosuppressive drugs rapamycin (RAPA) and cyclosporine A (CsA), and is probably related to inhibition of the 27‐hydroxylation of cholesterol (acid pathway of bile acid biosynthesis). This might be one of the causes for the increase in plasma cholesterol, as 27‐hydroxycholesterol is a potent suppressor of 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HMGR), a key enzyme of cholesterol synthesis. As the sterol 27‐hydroxylase (CYP27A1) inhibition by CsA is well known, we evaluated the effect of another immunosuppressive drug, RAPA, on this enzyme in HepG2 mitochondria, which confirmed the dose‐dependent inhibition of mitochondrial CYP27A1 by cyclosporine (10–20 µM), while the inhibition by RAPA required a higher dose (50–100 µM). Corresponding K_i was 10 µM for CsA (non‐competitive inhibition) and 110 µM for RAPA (competitive inhibition). Cotreatment with both immunosuppressive drugs showed an additive inhibitory effect on CYP27A1 activity. Later, we analysed the effect of these immunosuppressants on HMGR expression in HepG2 cells, and a dose‐dependent up‐regulation of HMGR gene expression was observed. The results suggest that RAPA and CsA are both inhibitors of CYP27A1 activity with slightly different mechanisms and that they may accordingly increase HMGR expression.