The reliability of accelerometry for measuring sedentary behavior in preschoolers has not been determined, thus we determined how many days of accelerometry monitoring are necessary to reliably estimate daily time spent in sedentary behavior in preschoolers. In total, 191 and 150 preschoolers (three to five years) wore ActiGraph accelerometers (15-s epoch) during the in-school (≥4 days) and the total-day (≥6 days) period respectively. Accelerometry data were summarized as time spent in sedentary behavior (min/h) using three different cutpoints developed for preschool-age children (<37.5, <200, and <373 counts/15 s). The intraclass correlations (ICCs) and Spearman-Brown prophecy formula were used to estimate the reliability of accelerometer for measuring sedentary behavior. Across different cutpoints, the ICCs ranged from 0.81 to 0.92 for in-school sedentary behavior, and from 0.75 to 0.81 for total-day sedentary behavior, respectively. To achieve an ICC of ≥0.8, two to four days or six to nine days of monitoring were needed for in-school sedentary behavior and total-day sedentary behavior, respectively. These findings provide important guidance for future research on sedentary behavior in preschool children using accelerometry. Understanding the reliability of accelerometry will facilitate the conduct of research designed to inform policies and practices aimed at reducing sedentary behavior in preschool children.