Kenaf nanofibers (NF) were isolated from the kenaf pulp using mechanical isolation methods (refining with super grounding and high pressure homogenization). The kenaf NF were acetylated to produce hydrophobic NF. FTIR results displayed a successful acetylation of the NF. X-ray analysis exhibited that the acetylation process reduced the crystallinity of kenaf NF but also that isolation to NF leads to higher crystallinity than corresponding micro-sized fibers. The contact angle measurements indicated that the acetylation treatment changed the surface characteristics of the kenaf NF from hydrophilic to more hydrophobic.