14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76-108 amino acids and the C-terminal 207-216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1-54 amino acids and the C-terminal 326-376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 → Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76-108 and C-terminal 207-216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1-54 or C-terminal 326-376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.