Objective The purpose of this study was to examine if parental separation during childhood is associated with obesity in adulthood. Methods A co-twin case-control study of 146 adult same-sexed twin pairs with discordant body mass index (BMI) (i.e. one of the twins should have a BMI of 20-25 kg/m(2), and the co-twin's BMI >= 30 kg/m(2)) was selected from Danish Twin Registry (DTR). In total of 236 eligible twin individuals participated in the study. Childhood parental separation (defined as separation from one of the biological parents, regardless of the reason for separation) for at least one year prior to age 17 was self-reported. The statistical analysis includes logistic and linear regression models using STATA 13.0. Results There were no differences in the odds of developing obesity in adulthood between the twin who stayed with a father and the co-twin who was separated from him for at least 1 year prior to age 17 [OR = 1.22, 95% CI (0.46-3.34), p = 0.65]. Separation from a mother also showed no differences in the odds for developing obesity [OR = 0.90, 95% CI (0.32-2.46), p = 0.82]. Conclusions Because of the limited number of discordant twin pairs for childhood parental separation, we cannot provide evidence to suggest that separation from parents in childhood was associated with developing obesity in adulthood. Further studies of pooling discordant twins from several countries should be considered.