In December, 2013, revised Alberta child care accreditation standards were released by the Alberta Government in Canada that included a new standard for physical activity and sedentary behavior in accredited child care settings. The main purpose of this study was to examine the effectiveness of the new accreditation standard in increasing physical activity and reducing sedentary behavior in a sample of children in Alberta, Canada attending child care. This pre-post design study is based on 86 children aged 19-60 months from across Alberta, Canada. Levels of physical activity [light (LPA) and moderate- to vigorous-intensity physical activity (MVPA)] and sedentary time during child care were accelerometer-derived at baseline (October/November, 2013) and follow-up after a new accreditation standard was implemented (May/June, 2014). Age- and sex-specific BMI z-scores at baseline and follow-up were also calculated. Changes in physical activity and sedentary behavior were moderated by age group (toddler: 19-35 months and preschoolers: 36-60 months). Over an average 6.5 month period, a small decrease in sedentary time (3.1 min/h, P < 0.05) and a moderate increase in MVPA (1.7 min/h, P < 0.05) were observed among toddlers along with a moderate decrease in BMI z-score (0.18 SD, P < 0.05). However, among preschoolers a small increase in sedentary time (1.9 min/h, P < 0.05) and a small decrease in LPA (1.9 min/h, P < 0.05) were observed. The new accreditation standard shows promise for toddlers. Future research with a control group that evaluates the process of implementing the new standard at each center is needed to confirm and expand on these findings of this study.