Carbon nanofibers (CNFs) with different content of surface functional groups which are carboxyl groups (CNF–OX), carbonyl groups (CNF–CO) and hydroxyl groups (CNF–OH) and nitrogen-containing groups (CNF–ON) are synthesized, and their electrocatalytic activities toward oxygen reduction reaction (ORR) in alkaline solution are investigated. The result of X-ray photoelectron spectroscopy (XPS) characterization indicates that a higher concentration of carboxyl groups, carbonyl groups and hydroxyl groups are imported onto the CNF–OX, CNF–CO and CNF–OH, respectively. Cyclic voltammetry shows that both the oxygen- and nitrogen-containing groups can improve the electrocatalytic activity of CNFs for ORR. The CNF–ON/GC electrode, which has nitrogen-containing groups, exhibits the highest current density of ORR. Rotating disk electrode (RDE) characterization shows that the oxygen reduction on CNF–ON/GC electrode proceeds almost entirely through the four-electron reduction pathway, the CNF–OX/GC, CNF–CO/GC and CNF–OH/GC electrodes proceed a two-electron reduction pathway at low potentials (−0.2 V to −0.6 V) followed by a gradual four-electron reduction pathway at more negative potentials, while the untreated carbon nanofiber (CNF–P/GC) electrode proceeds predominantly by a two-electron reduction pathway within the whole range of potential studied.