Gamma-glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K-dependent (VKD) proteins involved in a wide variety of physiological processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype-phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of three structure-function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter-proteins, then the carboxylation efficiency of these reporter-proteins were evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter-proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter-proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C-terminus plays a critical role in the binding of osteocalcin, but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein-protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre-mRNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes, and clarified why vitamin K administration rectified bleeding disorders but not non-bleeding disorders.