Phosphorylation of the Marburg virus nucleoprotein NP is distributed over 7 regions (I-VII) in its C-terminus. The exact localization of phosphorylated amino acids and function of NP phosphorylation are unknown. Here, we show that the major phosphate acceptor sites in NP region II are serine 446 and serines 453-455; the latter are located in a cluster of 6 serine residues (aa 450-455). The function of phosphorylation in region II was tested using an infectious virus-like particle assay. Phosphorylation influenced reporter gene activity that reflects viral transcription and replication. An NP mutant mimicking 3 phosphorylated serine residues at position 453-455 supported reporter gene activity better than wild-type NP. Negative charges at positions 450-452 and when the serine cluster was completely substituted by alanine inhibited reporter gene activity significantly. These data support the idea that phosphorylation of NP region II modulates viral RNA synthesis in transcription and/or replication.