The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS(1), MS(2) (neutral loss), MS(3) (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.