Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ-dependent mechanisms. Here we describe what we believe to be a novel IFN-γ-independent mechanism induced by β-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual β-linked sugar. Like α-galactosylceramide, β-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by β-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, β-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with β-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of β-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.