14-3-3 Proteins are a family of highly conserved regulatory molecules expressed in all eukaryotic cells and regulate a diverse set of biological responses in plants. However, their functions in flowering ofPhyllostachys violascensare poorly understood. In this study, four non-𝜀Pv14-3-3genes fromP. violascenswere identified and namedPvGF14b,PvGF14c,PvGF14e, andPvGF14f. qRT-PCR analyses revealed thatPvGF14bandPvGF14eexhibited widely expressed in all tested bamboo tissues.PvGF14bwas highest expression in root and lowest in immature leaf. WhereasPvGF14candPvGF14fshowed tissue-specific expression.PvGF14cwas mainly expressed in immature and mature leaves.PvGF14fwas highest expression in mature leaves. These four genes were not significantly differentially expressed in mature leaf before bamboo flowering and during flower development.PvGF14bandPvGF14cwere not induced by circadian rhythm. PvGF14c displayed subcellular localization in the cytoplasm and PvFT in nucleus and cytoplasm. Yeast two-hybrid screening and bimolecular fluorescence complementation confirmed the interaction between PvGF14c and PvFT. The overexpression ofPvGF14b,PvGF14c, andPvGF14esignificantly delayed flowering time in transgenicArabidopsisunder long-day condition. These findings suggested that at least threePvGF14genes are involved in flowering and may act as a negative regulator of flowering by interacting with PvFT in bamboo.