The objective of this study was to measure the mechanical properties of individual, native wood fibers using the continuous nanoindentation measurement technique. The indentation depth profile exhibited a small length-scale effect, which was confirmed using the size-effect index derived from the indentation loading curve. The hardness (Hu) or stiffness (Eu) values determined from indentation unloading were also examined for 10 different annual rings of a loblolly pine, with microfibril angles (MFA) between 14° and 36°. A predictable pattern of Eu values was found as a function of MFA, and hence Eu can at least be considered a relative measure of the longitudinal stiffness properties of wood cell walls. For Hu values, a dependence on orientation was observed, and there is a preliminary indication that the dependence could be affected by cell-wall extractives. It is thus desirable, for cell-wall modification studies, to minimize any unintended variations by using samples that are from the same growth ring, so that any treatment-induced changes in the cell-wall hardness can be identified.