Abstract The aim of this work was to evaluate the impact of atmospheric pressure nitrogen plasma on viability, proliferation, and osteogenic differentiation of normal mouse calvarial preosteoblasts (MC3T3-E1 Subclone 4), which were maintained in Hanks’ balanced salt solution (HBSS) during plasma exposure. Obtained results clearly demonstrated that short-time (4, 8, and 16 s) nitrogen plasma treatment is non-toxic to the MC3T3-E1 cells, does not affect cell morphology, promotes preosteoblasts’ proliferation, enhances osteogenic differentiation by increasing bone alkaline phosphatase and osteocalcin concentration, but inhibits mineralization of extracellular matrix. The best results were achieved for 16 s exposure time and when the preosteoblasts were left in HBSS for 3 h after plasma treatment. Presented studies indicate great clinical potential of cold atmospheric nitrogen plasma for regenerative medicine applications to improve bone healing process.