Rotavirus (RV) is the most important cause of severe gastroenteritis in children worldwide. Current live RV vaccines are efficacious but show lower efficacy in developing countries, as well as a low risk of intussusception. This has led to the development of parenteral non-live candidate vaccines against RV. RV capsid VP6 protein is highly conserved and the most abundant RV protein forming highly immunogenic oligomeric structures with multivalent antigen expression. Both recombinant VP6 (rVP6) or double-layered (dl) 2/6-virus-like particles (VLPs), might be considered as the simplest RV subunit vaccine candidates. Human rVP6 protein and dl2/6-VLPs were produced in Sf9 insect cells by baculovirus expression system. Formation of rVP6 tubules and VLPs were confirmed by electron microscopy. BALB/c mice were immunized intramuscularly, and immune responses were analyzed. Both rVP6 and dl2/6-VLPs induced a balanced Th1-type and Th2-type response and high levels of serum IgG antibodies with cross-reactivity against different RV strains (Wa, SC2, BrB, 69M, L26, WC3, and RRV). In addition, mucosal VP6-specific IgG and IgA antibodies were detected in feces and vaginal washes (VW) of immunized animals. Importantly, VWs of immunized mice inhibited RV Wa and RRV infection in vitro. Immunization with either protein preparation induced a similar level of VP6-specific, interferon-γ secreting CD4+ T cells in response to different RVs or the 18-mer peptide (AA242-259), a VP6-specific CD4+ T cell epitope. RV rVP6 and dl2/6-VLPs induced equally strong humoral and cellular responses against RV in mice and therefore, may be considered as non-live vaccine candidates against RV.