Cell-surface carbohydrate chains are known to contribute to cell migration, interaction, and proliferation. β-1,4-galactosyltransferase-I (β-1,4-GalT-I), which is one of the best-studied glycosyltransferases, plays a key role in the synthesis of type 2 chains in N-glycans and the core 2 branch in O-glycans. Recently, it has been reported that skin wound healing is significantly delayed in β-1,4-GalT-I−/− mice. However, the expression of β-1,4-GalT-I and its biological function in the skin wound-healing process remain to be elucidated. We used real-time polymerase chain reaction to demonstrate that the expression of β-1,4-GalT-I mRNA reached plateau values at 12 hours after skin was injured and remained elevated until 11 days after the injury. Furthermore, lectin blotting showed that β-1,4-galactosylated carbohydrate chains were also increased after skin injury. A double-staining method combining lectin-fluorescent staining with RCA-I and immunofluorescence was first used to determine the cellular localization of β-1,4-galactosylated carbohydrate chains. Morphological analysis showed that the chains were primarily expressed in neutrophils and partially expressed in macrophages, endothelial cells, and collagen. Our results suggest that β-1,4-GalT-I and β-1,4-galactosylated carbohydrate chains participate in leukocyte recruitment, angiogenesis, and collagen deposition in the skin wound-healing process.