Several factors affect the skeletal muscle differentiation process, in particular modifications of cell-cell contact, cell adhesion, and plasma membrane characteristics. In order to support the role of the plasma membrane-associated sialidase NEU3 in skeletal muscle differentiation and to analyse which events of the process are mainly affected by this sialidase, we decided to stably over-express MmNEU3 in C2C12 cells by a lentiviral vector and to investigate cell behavior during the differentiation process. Vitally stained C2C12 and NEU3 over-expressing cells were counted to reveal modifications in differentiation induction. We found that NEU3 over-expressing cells remained proliferative longer than control cells and delayed the onset of differentiation. Expression of p21, myogenic transcription factors, and myosin heavy chain (MHC), assessed by real time PCR, confirmed this behavior. In particular, no MHC-positive myotubes were present in NEU3 over-expressing cells as compared to wild type C2C12 cells at day 3 of differentiation. Moreover, NEU3 over-expressing cells completed the differentiation process very quickly and formed hypertrophic myotubes. Analysis of MAPK/ERK pathway activation showed an increased ERK 1/2 phosphorylation in NEU3 over-expressing cells at the beginning of differentiation. We postulate that sialidase NEU3, decreasing plasma membrane ganglioside GM3 content, affects the EGF receptor and the downstream signaling pathways, promoting proliferation and delaying differentiation. Furthermore NEU3 improves myoblast fusion probably via neural-cell adhesion molecule (NCAM) desialylation. Therefore, this work further supports the central role of NEU3 as a key modulator in skeletal muscle differentiation, particularly in the myoblast fusion step.