Rotavirus (RV) inner capsid VP6 protein is a potential non-live vaccine candidate due to high degree of conservation and immunogenicity, and ability to self-assemble into oligomeric structures, including nanotubes. These VP6 structures induce strong humoral and T cell immunity and protect mice against RV challenge. It has been suggested that intracellular neutralization by IgA antibody and VP6-specific CD4+ T cells mediate protection. We investigated generation of diverse CD4+ T cell subsets by intradermal and intranasal delivery of recombinant VP6 (rVP6) nanotubes in BALB/c mice. Production of antiviral cytokine interferon-γ (IFN-γ), interleukin-4 (IL-4) and pro-inflammatory cytokine IL-17 was analyzed following in vitro stimulation of immune cells. Cell surface CD107a expression was measured to determine VP6-specific cytotoxic T cells. Both parenteral and mucosal immunization with oligomeric rVP6 induced VP6-specific Th1, Th2 and Th17 cells. For the first time, cytotoxicity-related degranulation (CD107a surface expression) indicated that RV VP6-specific CD4+ T cells had cytotoxic T lymphocyte (CTL) phenotype. These findings demonstrate an ability of rVP6 nanostructures to induce heterogeneous CD4+ T cells with different effector functions, including CTLs with potential to lyse RV-infected cells, suggesting an additional mechanism of RV VP6-induced protection.