This study aimed to evaluate by microCT the preparation of the artificial teeth (ATs), TrueTooth®, versus natural teeth (NTs): (1) the time and number of pecking movements needed for preparation; (2) the root canal volume increase; (3) if the pulp-colored medium has any effect on the 3D analysis. Material and Methods: Artificial and natural maxillary molars were used. Fourteen AT distobuccal canals and fourteen NT buccal canals were used for the first and second aim and fourteen AT mesiobuccal canals for the third aim. Results: No statistically significant differences were observed regarding the time and number of pecking movements (p > 0.05); for the root canal volume increase, a statistically significant difference was observed (p < 0.05) with a higher mean value for NTs; however, in the group of ATs, there was a volume decrease in three cases. The AT mesiobuccal root canal mean volume increase was also negative. Conclusions: There are no differences between the time and number of pecking movements between NTs and ATs, so TrueTooth® can potentially be used in endodontic training. The volume increase between ATs and NTs was higher in NTs. However, some samples showed negative values, also seen in the AT mesiobuccal canal, confirming that the pulp-colored medium has an effect on the 3D analysis.