Site-specific mutants as candidates for live influenza vaccines were resulted from directly introducing into the genome of the pathogenic influenza virus A/WSN/33 (H1N1) strain ts mutations derived from the genes encoding the polymerase complex proteins from some cold-adapted strains serving as attenuation donor. Here we present the data of a comparative study examining immune system arms in mice immunized intranasally with influenza virus mutants and classical cold-adapted reassortant obtained by crossing cold-adapted strain Donor A/Krasnodar/101/35/59 (H2N2) with strain A/WSN/33 (H1N1) bearing surface antigens (hemagglutinin and neuraminidase) similar to mutants. Immunophenotyping mononuclear leukocytes from immunized mice indicated at moderate suppressive effect after using site-specific mutant and the HA reassortant viruses on some immune cell subsets. All viruses in immunized mice resulted in activation of certain lymphocyte subsets including MHC II-positive cells, CD45+/CD19+ B lymphocytes and natural killer cells (CD16/32+/CD3–). Timescale and magnitude of activation markedly differed for each cell subsets. Mice immunized with mutants M26 and U2 peaked with count of CD16/32+/CD3–expressing cells on day 2 after the second immunization compared with control (p < 0.05) that may suggest about an important role for NK cells in activating immune response. In contrast, no significant changes were observed during the study in percentage of CD4+/CD25+/Fox P3 regulatory T cells, CD4+ T helpers and CD8+ cytotoxic cells, except for a sharply decreased count of activated CD4+/CD25+ cells (4-fold) on day 7 after immunization with mutant virus M26. Moreover, mutants U2 and M26 more moderately increased percentage of TLR2- and TLR4-positive cells. The viruses studied ambiguously affected count of TLR9-expressing cells in immunized animals. All viruses increased phagocytic activity in monocytes, but not neutrophils. Despite the moderate activation of innate and adaptive immunity arms, site-specific mutants more profoundly affected humoral reactions inducing increased antibody titers, so that immunogenicity of mutant viruses was higher than that of the cold-adapted reassortant. Thus, the findings hold a promise of using site-specific mutants as live influenza vaccines. © 2020 Saint Petersburg Pasteur Institute. All rights reserved.