Formyl peptide receptor 2 (FPR2), a G protein‐coupled receptor (GPCR), transduces both proinflammatory and anti‐inflammatory signals in an agonist‐dependent manner. To determine how a GPCR transduces opposing signals, fluorescent biosensors of FPR2 were constructed using single‐molecule Frost resonance energy transfer (FRET). Incubated with Ac2‐26, an annexin AI‐derived peptide ligand with anti‐inflammatory activity, induced decreases in FRET intensity at 10 pM that is far below its Ca2+ flux‐inducing concentrations. WKYMVm (W‐pep) and Aβ42, both proinflammatory agonists of FPR2, stimulated increases in FRET intensity, suggesting that different FPR2 conformational states were induced for pro‐ and anti‐inflammatory signaling. Ac2‐26 and Aβ42 produced biphasic dose curves, but in opposite directions. Neither ligands could compete for W‐pep binding at this concentration, suggesting that they use allosteric sites on FPR2. Preincubation with Ac2‐26 at 10 pM diminished subsequent response to W‐pep in Ca2+ and cAMP inhibition assays, reduced FPR2 binding of W‐pep and ERK phosphorylation, but potentiated W‐pep‐stimulated membrane translocation of β‐arrestin2 and phosphorylation of p38 MAPK. The opposite effects were observed when the FPR2‐expressing cells were first incubated with 10 pM of Aβ42 and then stimulated with W‐pep. These results identified Ac2‐26 and Aβ42 as biased allosteric modulators at the subnanomolar concentrations used, that dose‐dependently alter signaling of the orthosteric agonist WKYMVm. Exploration of this feature may help direct FPR2 to pro‐ or anti‐inflammatory signaling pathways.Support or Funding InformationThis work was supported in part by the National Natural Science Foundation of China grant 31470865.