Background: Infection with hepatitis C virus (HCV) lowers serum cholesterol levels, which rapidly recover during therapy with direct-acting antivirals (DAAs). Serum cholesterol is also reduced in patients with liver cirrhosis. Studies investigating serum cholesterol in patients with chronic liver diseases are generally based on enzymatic assays providing total cholesterol levels. Hence, these studies do not account for the individual cholesteryl ester (CE) species, which have different properties according to acyl chain length and desaturation. Methods: Free cholesterol (FC) and 15 CE species were quantified by flow injection analysis high-resolution Fourier Transform mass spectrometry (FIA-FTMS) in the serum of 178 patients with chronic HCV before therapy and during treatment with DAAs. Results: Serum CEs were low in HCV patients with liver cirrhosis and, compared to patients without cirrhosis, proportions of CE 16:0 and 16:1 were higher whereas % CE 20:4 and 20:5 were reduced. FC levels were unchanged, and the CE/FC ratio was consequently low in cirrhosis. FC and CEs did not correlate with viral load. Four CE species were reduced in genotype 3 compared to genotype 1-infected patients. During DAA therapy, 9 of the 15 measured CE species, and the CE/FC ratio, increased. Relative to total CE levels, % CE 16:0 declined and % CE 18:3 was higher at therapy end. At this time, % CE 14:0, 16:0 and 16:1 were higher and % CE 20:4 and 22:6 were lower in the cirrhosis than the non-cirrhosis patients. Viral genotype associated changes of CEs disappeared at therapy end. Conclusions: The serum CE composition differs between patients with and without liver cirrhosis, and changes through the efficient elimination of HCV. Overall, HCV infection and cirrhosis are associated with a higher proportion of CE species with a lower number of carbon atoms and double bonds, reflecting a less-favorable CE profile.