Hemp fibers are broadly used in industrial applications for their mechanical properties, in particular their Young's modulus (YM) that is comparable to glass fibers, and for their low density, which makes them suitable for environmentally friendly alternatives to petrochemical compounds in applications such as in construction, bioplastics, or composite materials. However, their sensitivity to humidity can impact the properties of materials produced. Here, the effect of relative humidity (RH) in air on the local indentation modulus of hemp fibers was explored at the nanoscale so as to differentiate the behavior of individual cell wall layers. A decrease in the indentation modulus of xylem and sclerenchyma fibers with increasing RH was unveiled and quantified using atomic force microscopy. The nanoscale analysis revealed that xylem and sclerenchyma behave differently under increasing RH, suggesting that the macromolecular architecture of the cell walls impacts the interaction with water molecules. The moisture content, measured using dynamic vapor sorption, indicates higher water content in xylem fibers than in sclerenchyma fibers during the sorption phase. The differences in lignin content and crystallinity of cellulose were assessed with Raman spectroscopy, further underlying the role the composition of plant fibers plays in water uptake. Overall, the study deepens the understanding of the interplay between composition and moisture and their effect on nanomechanical properties of hemp and hemp-based composites, signifying that materials derived from hemp bast fibers will exhibit more stable hygroscopic behavior.