Nucleolin is an ubiquitous, nonhistone nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation, and growth. Nucleolin was primarily found in the nucleus, but it was also proposed as a possible shuttle between the nucleus, cytoplasm, and cell membrane. We report here that part of the extranuclear nucleolin undergoes complex N- and O-glycosylations. A band with higher molecular mass (113 kDa) than the 105-kDa classical major nucleolin band was detected on SDS-PAGE gel that cross-reacted with specific anti-nucleolin antibodies and was identified as a nucleolin isoform by mass spectrometry. The presence of N-glycans was first suggested by sensibility of the 113-kDa nucleolin isoform to tunicamycin treatment. Determination of monosaccharide composition by heptafluorobutyrate derivation followed by gas-chromatography mass spectrometry indicated the presence of N- and O-glycans. The structures of N- and O-glycans were first investigated using specificity of binding to lectins. This approach allowed a partial characterization of N-glycan structures and revealed O-glycan structures that could otherwise go unnoticed. Further study of N-glycans by mass spectrometry using direct exoglycosidase treatment on MALDI-TOF target allowed the complete definition of their structures. Finally, the use of peptide mass fingerprinting with sinapinic acid allowed identification of N317 and N492 as the two N-glycosylation sites. N317 and N492 belong to RNA-binding domains 1 and 3 of nucleolin, respectively, that suggests a role of glycosylation in regulating the function of the protein.