In this study, the effect of pectin extraction method on the properties of cellulose nanofibers (CNFs) isolated from sugar beet pulp (SBP) was studied. Pectin was extracted by the industrially practiced method by sulfuric acid hydrolysis or by enzymatic hydrolysis using a cellulase/xylanase enzymes mixture. The CNFs were then isolated by high-pressure homogenization and investigated in terms of their chemical composition, crystallinity, size, degree of polymerization, and re-dispersion in water after freeze-drying. The mechanical properties and surface characteristics of CNF films were also studied. The results showed that fibrillation of the de-pectinated SBP was more efficient for the acid hydrolyzed SBP. CNFs from the acid-hydrolyzed SBP had a slightly wider diameter, higher crystallinity, viscosity, and α-cellulose content but a lower degree of polymerization than CNFs from the enzyme-hydrolyzed SBP. Owing to the presence of more residual hemicelluloses in the CNFs from the enzyme-hydrolyzed SBP, the CNFs had higher re-dispersion ability in water. CNF films from enzyme-hydrolyzed SBP displayed slightly better mechanical properties and higher water contact angle than acid-hydrolyzed CNF films.