Assessment of cancellous bone connectivity has the potential to aid in predicting fracture risk. Today, cancellous bone connectivity is generally assessed using bone sections obtained from biopsy. However, how reliably such two-dimensional (2-D) analyses visualize the 3-D properties has not been evaluated. Biopsied iliac bone samples were obtained from 47 chronic hemodialysis patients. Bone samples were observed using a microfocus X-ray computed tomography (microCT) system en bloc, and the cancellous bone microstructure was quantitatively assessed at both the 2- and 3-D levels. Cancellous bone microarchitecture was successfully reconstructed from the data obtained by the microCT system. Most of the results from node-strut analysis (NSA) revealed no statistically significant correlations between the 2- and 3-D analyses, with the exception that the number of nodes (N.Nd/TV) showed a mild but significant correlation. In contrast, the marrow space star volumes (V*m) of the 2- and 3-D analyses were highly correlated. NSA parameters including N.Nd/TV showed significant correlations with V*m at the 3-D level. In conclusion, V*m values were similar in the 2- and 3-D analyses, while most of the 2-D NSA parameters did not reflect the 3-D ones. Since V*m and most of the NSA parameters were correlated in the 3-D analyses, 2-D NSA would seem to have serious limitations for the assessment of cancellous bone microstructural properties. Further studies will thus be needed to establish appropriate methods for assessing cancellous bone connectivity in clinical practice.