The glucose-6-phosphate transporter (G6PT) deficient in glycogen storage disease type Ib is a phosphate (P(i))-linked antiporter capable of G6P: P(i) and P(i):P(i) exchanges. We previously characterized G6PT mutations by measuring G6P uptake activities in microsomes co-expressing G6PT and glucose-6-phosphatase-alpha. Here we report a new assay, based on reconstituted proteoliposomes carrying only G6PT, and characterize G6P and P(i) uptake activities of 23 G6PT mutations. We show that co-expression and G6PT-only assays are equivalent in measuring G6PT activity. However, the p.Q133P mutation exhibits differential G6P and P(i) transport activities, suggesting that characterizing G6P and P(i) transport activities of G6PT mutations may yield insights to this genetic disorder.