Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.