The quality of apical seal with regard to the length of remaining gutta-percha following post-space preparation is still controversial. The purpose of this in vitro study was to compare three different lengths of remaining gutta-percha for apical seal after post-space preparation. A total of 126 single-rooted extracted maxillary human anterior teeth with intact apices, straight roots, and without resorption were used in this study. The root canals were prepared and filled with gutta-percha and AH26 sealer. The post-space preparation was accomplished. Ninety-six teeth were randomly divided into three groups (4, 5 and 6 mm of gutta-percha was retained in group 1, 2 and 3, respectively).Thirty teeth were considered for the control groups in which 5 teeth served as positive and 5 teeth served as negative controls. The specimens were placed in India ink for 48 hours and then divided into two halves. The amount of leakage was observed and measured with a stereomicroscope at x 16 magnification and 0.1 mm accuracy. The results showed that there were significant differences among the three experimental groups (P < 0.05). The best apical seal after post-space preparation was associated with the maximum length of remaining gutta-percha in the apical portion of the treated teeth.