Study the effect of root canal preparation in three different tapers on the effect of root canal filling using the second generation novel cold flow root canal filling system in vitro. 60 extracted human premolars with single root canal were divided randomly into 3 groups(A:02taper, B:04taper, C:06taper and N=20). The root canals were prepared using NIC® Super File to 3 different tapers to get to 30#,and were obturated with GuttaFlow2 in single–cone obturationtechnique.Then the roots were sectioned at 3 and 6mm from apical foramen.All the sections were observed under a stereo–microscope.Then these images were collected and analyzed.The percentage of gutta–percha–filled area(PGFA) was calculated by the software.The filling effect of root canals were evaluated and recorded by X–ray. As the increasing of the root canal taper,at the 3mm level from apex, the PGFA of group C was significantly higher than that of the group A (p<0.05),and the PGFA between the three groups was statistical significance (P<0.05). At the 6mm level from apex,those of 3 groups have no statistical significance (p>0.05). As the increasing of taper, the number of under–filled root canal in the 3 groups decreased, but there was no statistical significance in the 3 groups. When the taper of NI–TI instrument increase up to 06, it can effectively improve the effect of sealing in root canal filling and reduce the under–filled with the GuttaFlow2.But the effect of resisting force of the tooth root remains to be studied.