The aim of this study was to investigate the suitability of yeast Saccharomyces cerevisiae expression system for the production of human parainfluenza virus type 2 (HPIV2) nucleocapsid (N) protein in the form of nucleocapsid-like particles (NLPs) and to characterize its antigenic structure. The gene encoding HPIV2 N amino acid (aa) sequence RefSeq NP_598401.1 was cloned into the galactose-inducible S. cerevisiae expression vector and its high-level expression was achieved. However, this recombinant HPIV2 N protein did not form NLPs. The PCR mutagenesis was carried out to change the encoded aa residues to the ones conserved across HPIV2 isolates. Synthesis of the modified proteins in yeast demonstrated that the single aa substitution NP_598401.1:p.D331V was sufficient for the self-assembly of NLPs. The significance of certain aa residues in this position was confirmed by analysing HPIV2 N protein structure models. To characterize the antigenic structure of NLP-forming HPIV2 N protein, a panel of monoclonal antibodies (MAbs) was generated. The majority of the MAbs raised against the recombinant NLPs recognized HPIV2-infected cells suggesting the antigenic similarity between the recombinant and virus-derived HPIV2 N protein. Fine epitope mapping revealed the C-terminal part (aa 386-504) as the main antigenic region of the HPIV2 N protein. In conclusion, the current study provides new data on the impact of HPIV2 N protein sequence variants on the NLP self-assembly and demonstrates an efficient production of recombinant HPIV2 N protein in the form of NLPs.