Guided endodontics has various applications, one of which is for calcified canal negotiation. Recently, a new single-tooth template has been fabricated to overcome the drawbacks of bulky guides, which are difficult to use with rubber dam isolation.This study aimed to assess the efficacy of the novel single-tooth template for negotiation of pulp canal calcification (PCC) in three-dimensional (3D)-printed resin incisors by comparing substance loss and time taken between incisal endodontic access (IEA) and single-tooth template-guided endodontic access (SGEA).Forty-two resin incisor teeth having patent canal in the apical third were used (N = 21/group). They were subcategorized based on operator's experience into senior endodontist (SE), postgraduate (PG), and undergraduate (UG) (n = 7/operator). Canals were negotiated conventionally for IEA and using the single-tooth template for SGEA. Substance loss was calculated from the volume difference between pre- and postoperative cone-beam computed tomography scans. The time taken was also recorded.Statistical analysis was performed using unpaired t-test and one-way analysis of variance test.Canals were successfully negotiated in 100% and 95% of teeth in the SGEA and IEA groups, respectively. Overall substance loss and time taken were significantly lesser for SGEA for all operators (P < 0.001). In the IEA group, post hoc test showed statistical significance between SE and UG for substance loss (P < 0.05) and SE-UG and PG-UG for time taken (P < 0.05). No significant difference among operators was noted for both parameters in SGEA.SGEA resulted in significantly lesser substance loss and time taken for canal negotiation in 3D-printed resin incisors with simulated PCC. This was independent of the experience levels of the operator.