Human Ero1-Lalpha catalyzes the formation of disulfide bond and hence plays an essential role in protein folding. Understanding the mechanism of disulfide bond formation in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. However, the crystal structure of the enzyme is not available yet, which seriously hinders the understanding of biological function of Ero1-Lalpha. Based on the crystal structure of yeast Ero1p, a rational three-dimensional structural model of Ero1-Lalpha was built and the characteristics of the enzyme were hence investigated. The characteristic similarities and differences between Ero1-Lalpha and Ero1p were compared on the basis of computational and experimental results, providing the first insight into the structure-function relationships of the enzymes. Both calculation and experiment got the concordant conclusion that FAD binds more tightly with Ero1-Lalpha than Ero1p. In addition, the probable electron transfer pathway was proposed on the basis of the structural models.