NKp80, also known as KLRF1, is an activating homodimeric C-type lectin-like receptor that is expressed on nearly all-natural killer cells and stimulates their cytotoxicity and cytokine release. NKp80 stimulates cytotoxicity upon engagement of its genetically linked ligand: myeloid-specific CTLR activation-induced C-type lectin (AICL). NKp80, but not NKp80 mutated at tyrosine 7 (NKp80/Y7F), is tyrosine phosphorylated. Accordingly, NKp80/Y7F, but not NKp80/Y3F or NKp80/Y37F, failed to induce cytotoxicity. NKp80 phosphopeptides comprising the Hemi-ITAM-like sequence surrounding tyrosine 7 bound Lck- and Syk-family kinases; accordingly, cross-linking of NKp8, but not NKp80/Y7F, induced Syk phosphorylation. Moreover, inhibition of Syk kinase, but not ZAP-7 kinase, impaired cytotoxic responses through NKp80. Atypical residues in the Hemi-ITAM-like motif of NKp80 cause an altered stoichiometry of phosphorylation but did not substantially affect NK cytotoxicity. Altogether, these results show that NKp80 uses an atypical Hemi-ITAM and Syk kinase to trigger cellular cytotoxicity.