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Aims/Introduction To observe the longitudinal changes in serum adipocyte fatty acid-binding protein ( AFABP), carbohydrate, and lipid metabolism parameters in women with and without gestational diabetes mellitus ( GDM) during mid- and late pregnancy periods, as well as to identify whether there is any association between AFABP and development of GDM. Materials and Methods A total of 40 GDM and 240 normal glucose tolerance participants were enrolled at 24-28 weeks and completed the study. The clinical features, serum AFABP, other adipocytokines (leptin, adiponectin, retinol-binding protein 4), homeostasis model assessment of insulin resistance, and lipid profiles were measured in the second and third trimesters of pregnancy. Results Compared with the normal glucose tolerance group, the GDM group showed greater levels of AFABP, leptin and retinol-binding protein 4; and a decreased level of adiponectin ( P < 0.05 or P < 0.01) during mid- and late pregnancy periods. Prepregnancy body mass index was the independent factor impacting serum AFABP levels in the second (β = 0.567, P = 0.004) and third trimesters (β = 0.619, P = 0.001). Furthermore, GDM was independently associated with AFABP concentrations in multiple regression analysis in the second and third trimester (all P < 0.01). Serum AFABP, leptin and retinol-binding protein 4 are risk factors for GDM; adiponectin is a protective factor for GDM ( P < 0.05 or P < 0.01). Conclusions The GDM group had a higher level of AFABP during mid- and late stages of pregnancy; prepregnancy body mass index and GDM were the independent factors with respect to serum AFABP. AFABP might be closely related to obesity, insulin resistance and leptin resistance in pregnancy, and is a major risk factor for GDM. [ABSTRACT FROM AUTHOR]