Abstract Objectives Parental self-care is extremely important in the face of stress throughout parenthood. A 21-day online mindfulness-based intervention was developed that was aimed at enhancing parental well-being. The present study evaluated this intervention by examining its initial efficacy on parents’ mindfulness, parenting stress, subjective well-being, and symptoms of depression and anxiety. Methods Participants were 273 parents (90.11% mothers) who were randomly assigned to the 21-day mindfulness-based intervention group (n = 136) or waitlist control group (n = 137). Pre-intervention assessment, immediate post-intervention assessment, and 30-day follow-up assessment were conducted to assess parents’ mindfulness, parenting stress, subjective well-being, and symptoms of depression and anxiety. Results Linear mixed models indicated that the group × time effects on subjective well-being, anxiety symptoms, and mindfulness were significant, after controlling for sex, age, education, income, habit of mindfulness practice, hours of weekly mindfulness practice, and diagnostic history of psychiatric disorder. Follow-up analyses indicated that compared to baseline, participants from the intervention group reported significantly greater subjective well-being and mindfulness, and fewer symptoms of anxiety than did those from the waitlist control group. The group × time effects on parenting stress and depressive symptoms were non-significant. Exploratory findings further suggested practicality and perceived acceptability of the intervention. Conclusions This study showed initial efficacy of a 21-day online mindfulness-based intervention on parents’ subjective well-being, anxiety symptoms, and mindfulness. The findings inform researchers and practitioners about the utility of a brief mindfulness-based intervention in promotion parental well-being. Other areas of feasibility warrant future investigation.