The aim of this study was to investigate the effects of GCKR polymorphism on the prevalence of overweight and obesity in Chinese female subjects using a gene-wide tagging- single nucleotide polymorphism (tSNP) strategy. We conducted a genetic association study in the Taizhou Retiree Women Cohort, a sub-cohort of the Taizhou longitudinal study. We genotyped four tSNPs (rs4425043, rs780094, rs814295, and rs8179206) of the GCKR gene using the Taqman assay in 2,851 female subjects and investigated their associations with overweight and obesity. Odds ratios and their 95% confidence intervals (CIs) were derived from ordered logistic regression model. We observed significant association between rs4425043 and body-mass-index-defined overweight and obesity. The frequencies of A allele of the rs4425043 exhibited a significant increasing trend from normal weight (13.20%), overweight (15.08%), to obese subjects (17.10%) (P = 0.006). Individuals with the GA or AA genotypes showed a 31% excessive risk to develop overweight or obesity (95% CI: 1.12-1.52, P = 0.001). In addition, we observed significantly increased levels of fasting plasma glucose associated with variations of both rs780094 and rs814295 (5.03, 5.09, and 5.15 mmol/L for rs780094 AA, GA and GG genotypes, respectively, and 5.03, 5.11, and 5.20 mmol/L for rs814295 AA, GA and GG genotypes, respectively). In conclusion, a novel polymorphism (rs4425043) in the GCKR gene increases the risk of overweight and obesity in Chinese women. Previous report that other polymorphisms in the GCKR gene are associated with glucose levels have also been confirmed.