The study objective was to determine cementum-dentin wall thickness along the furcation groove in maxillary bifurcated first premolars after preparation with three successively larger, 0.04 tapered, nickel titanium rotary files. Pre-instrumentation and post-instrumentation imaging was accomplished utilizing Cone Beam Computed Tomography. All data was analyzed using an ANOVA. Instrumentation resulted in a significant reduction in dentin-cementum wall thickness (p < .001). At mid-groove, predicted dentin-cementum wall thickness (95% CI) was equal to or less than the proposed standard (0.50 mm) for apical file sizes 30, 35, and 40 respectively. Instrumentation of the mid-groove in maxillary first premolars reduces dentin-cementum wall thickness to levels that may be insufficient to ensure tooth integrity.