To improve the applicability of the toxic equivalents principle for human health risk assessment, systemic relative effect potencies (REPs) for dioxin-like compounds (DLCs) deriving from human in vivo data are required. A prospective nested case-control study was performed to determine REPs from the human serum concentration of DLCs using gestational diabetes mellitus (GDM) and fasting blood glucose (FBG) as the end points of concern. Serum concentration of 29 DLCs from 77 cases and 154 controls were measured. Logistic and linear regression were used to estimate the effects of individual congeners on GDM and FBG, respectively. The REPs based on GDM and FBG were calculated from the ratios of regression coefficients, βi (DLCs)/βTCDD. Two sets of consistent human serum-based REPs, that is, GDM-REP and FBG-REP, were established and largely agree with REPs from other human studies. These human-serum REPs show much smaller variation compared to the 4 to 5 orders of magnitude span in REPs database for the present WHO-TEF determination. Moreover, the established REPs fitted well with WHO-TEFs, especially for polychlorinated dibenzo- p-dioxins, furans. These REPs reflecting real human exposure scenarios exhibited validity and could be used to improve health risk assessment of human body burden of DLCs.