Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen pMLL 747-755 (EPR(pS)PSHSM) is recognized by cognate TCR27, which is a candidate for immunotherapy of AML. We show that the replacement of phosphoserine P 4 with serine or phosphomimetics does not affect the pMHC conformation or peptide-MHC affinity but abrogates the TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. We determined the crystal structures for TCR27 and cognate pMHC, mapped the pMHC-TCR interface by TROSY-NMR, generated a ternary pMHC-TCR complex using information-driven protein docking, and identified key polar interactions between phosphate group at P 4 and TCR27 that are crucial for ternary complex stability and TCR27 specificity. These data will support development of cancer immunotherapy through target expansion and TCR optimization. *The authors would like to note that Yury Patskovsky and Aswin Natarajan contributed equally.