The aim was to develop a standardized curved root canal model in bovine dentine and to assess whether that natural substrate would behave differently from the resin in standard plastic training blocks when prepared chemo-mechanically. The impact of substrate microhardness on simulated canal transportation was considered. High-precision computer numerical control (CNC) milling was used to recreate a simulated root canal from a resin training block (Endo Training Bloc J-Shape, size 15) in longitudinally sectioned, dis- and re-assembled bovine incisor roots. Optical overlays obtained from 10 resin blocks were used to identify an average canal and program the CNC milling apparatus accordingly. Resin and dentine microhardness were measured. Simulated root canals in resin training blocks and their bovine counterparts were then instrumented at 37°C using Reciproc R25 instruments (VDW) with water or 17% EDTA (n = 10). Open-access image processing software was used to superimpose and analyse pre- and postoperative images obtained with a digital microscope. Centering ratios were averaged to indicate canal transportation. The effects of substrate and irrigant on canal transportation were assessed by two-way anova. Superimposed images showed that resin blocks under investigation varied considerably in terms of simulated canal length and curvature, whilst the milled canals were highly similar. The microhardness of dentine was more than three times higher than that of the resin. Conversely, canal transportation was considerably greater in dentine compared to resin, and in dentine had a tendency to be increased by EDTA. There was a strong effect of substrate on canal transportation (p < .001), no overall effect of irrigant, and a marginally significant interaction between irrigant and substrate (p = .077). CNC milling allows to create standardized simulated curved root canals in bovine dentine. These models may be useful to test and compare materials and concepts of chemo-mechanical root canal instrumentation. Microhardness is a bulk feature that does not predict the response to chemo-mechanical instrumentation of a composite material such as dentine.