Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two‑dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.