Combined deficiency of factors V and VIII (F5F8D) is a bleeding disorder caused by mutations in LMAN1 or MCFD2. LMAN1 encodes ERGIC-53, a cargo receptor with an L-type lectin domain, and MCFD2 is a EF-hand-containing protein. We prepared a biotinylated, soluble form of ERGIC-53, which we labeled with R-phycoerythrin conjugated streptavidin. By flow cytometry, sERGIC-53-SA bound to HeLaS3 cells in the presence of calcium but only after preincubation with MCFD2. Treating the cells with endo H or incubating them with high mannose-type oligosaccharides, especially M(8B), abrogated sERGIC-53-SA binding. Surface plasmon resonance experiments demonstrated that MCFD2 specifically bound to sERGIC-53 and 2 MCFD2 mutants found in F5F8D patients had a K(a) that was 3 or 4 orders of magnitude lower for sERGIC-53 than for wild-type MCFD2. The K(a) of sERGIC-53 and MCFD2 was measured at several pH values and calcium concentrations, and we found that at a calcium concentration less than 0.2 mM, this interaction became significantly weaker. These results demonstrate that the binding of ERGIC-53 to sugar is enhanced by its interaction with MCFD2, and defects in this interaction in F5F8D patients may be the cause for reduced secretion of factors V and VIII.