The objective of this study was to identify proteins in the m. longissimus dorsi between early (12 months of age) and late (27 months of age) fattening stages of Hanwoo (Korean cattle) steers. Using two-dimensional electrophoresis and mass spectrometry, 8 proteins of 11 differentially expressed spots between the 12 and 27 month age groups were identified in the loin muscle. Among those that were differentially expressed, zinc finger 323 and myosin light chain were highly expressed in late-fattening stage, and two catabolic enzymes, triosephosphate isomerase (TPI) and succinate dehydrogenase (SDH) were expressed more in the early versus the late-fattening stage. In particular, the quantification of TPI and SDH by immunoblotting correlated well with fat content. Our data suggested that TPI and SDH are potential candidates as markers and their identification provides new insight into the molecular mechanisms and pathways associated with intramuscular fat contents of bovine skeletal muscle.