Objectives Parenting is an integral component of obesity treatment in early childhood. However, the link between specific parenting practices and treatment effectiveness remains unclear. This paper introduces and validates a new parenting questionnaire and evaluates mothers’ and fathers’ parenting practices in relation to child weight status during a 12-month childhood obesity treatment trial. Methods First, a merged school/clinical sample (n = 558, 82% mothers) was used for the factorial and construct validation of the new parenting questionnaire. Second, changes in parenting were evaluated using clinical data from the More and Less Study, a randomized controlled trial (RCT) with 174 children (mean age = 5 years, mean Body Mass Index Standard Deviation Score (BMI SDS) = 3.0) comparing a parent support program (with and without booster sessions) and standard treatment. Data were collected at four time points over 12 months. We used linear mixed models and mediation models to investigate associations between changes in parenting practices and treatment effects. Findings The validation of the questionnaire (9 items; responses on a 5-point Likert scale) revealed two dimensions of parenting (Cronbach’s alpha ≥0.7): setting limits to the child and regulating one’s own emotions when interacting with the child, both of which correlated with feeding practices and parental self-efficacy. We administered the questionnaire to the RCT participants. Fathers in standard treatment increased their emotional regulation compared to fathers in the parenting program (p = 0.03). Mothers increased their limit-setting regardless of treatment allocation (p = 0.01). No treatment effect was found on child weight status through changes in parenting practices. Conclusion Taken together, the findings demonstrate that the new questionnaire assessing parenting practices proved valid in a 12-month childhood obesity trial. During treatment, paternal and maternal parenting practices followed different trajectories, though they did not mediate treatment effects on child weight status. Future research should address the pathways whereby maternal and paternal parenting practices affect treatment outcomes, such as child eating behaviors and weight status.