Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.