The purpose of this systematic review was to determine whether smear layer removal reduces leakage of obturated human teeth in vitro. PubMed was searched for articles published between 1975 and 2005, and results were categorized based on the method of leakage test. Among 26 eligible papers with 65 comparisons, 53.8% of the comparisons reported no significant difference, 41.5% reported a difference in favor of removing the smear layer, and 4.7% reported a difference in favor of keeping it; differences were significant (p<0.001). Of the 65 comparisons, 44 used the dye leakage test for evaluation. The combined effect in this group showed smear layer removal decreases dye leakage (z-score=0.37, z=2.31, p=0.021). According to meta-regression, obturation type, test site and duration, sealer and dye, and publication year had no effect on the results. Under the conditions of these in vitro leakage studies, it is concluded that smear layer removal improves the fluid-tight seal of the root canal system whereas other factors such as the obturation technique or the sealer, did not produce significant effects.