The liver is the major contributor to homocysteine (Hcy) metabolism and fatty liver disease is associated with hyperhomocysteinemia. Bleomycin hydrolase (Blmh) is an aminohydrolase that also participates in Hcy metabolism by hydrolyzing Hcy-thiolactone. To gain insight into hepatic functions of Blmh, we analyzed the liver proteome of Blmh(-/-) and Blmh(+/+) mice in the absence and presence of diet-induced (high methionine) hyperhomocysteinemia using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We identified eleven liver proteins whose expression was significantly altered as a result of the Blmh gene inactivation. The differential expression (Blmh(-/-) vs. Blmh(+/+)) of four liver proteins was lower, of two proteins was higher, and was further modified in mice fed with a hyperhomocysteinemic high-Met diet. The down-regulated proteins are involved in lipoprotein metabolism (ApoA1, ApoE), antigen processing (Psme1), energy metabolism (Atp5h, Gamt), methylglyoxal detoxification (Glo1), oxidative stress response (Sod1), and inactivation of catecholamine neurotransmitters (Comt). The two up-regulated proteins are involved in nitric oxide generation (Ddah1) and xenobiotic detoxification (Sult1c1). We also found that livers of Blmh(-/-) mice expressed a novel variant of glyoxalase domain-containing protein 4 (Glod4) by a post-transcriptional mechanism. Our findings suggest that Blmh interacts with diverse cellular processes-from lipoprotein metabolism, nitric oxide regulation, antigen processing, and energy metabolism to detoxification and antioxidant defenses-that are essential for liver homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in fatty liver disease.