Bioinformatic analysis indicates that sucrose phosphate synthase (SPS) contains a putative C-terminal sucrose phosphate phosphatase (SPP)-like domain that may facilitates the binding of SPP. If an SPS-SPP enzyme complex exists, it may provide sucrose biosynthesis with an additional level of regulation, forming a direct metabolic channel for sucrose-6-phosphate between these two enzymes. Herein, the formation of an enzyme complex between SPS and SPP was examined, and the results from yeast two-hybrid experiments suggest that there is indeed an association between these proteins. In addition, in planta bioluminescence resonance energy transfer (BRET) was observed in Arabidopsis seedlings, providing physical evidence for a protein interaction in live cells and in real time. Finally, bimolecular fluorescence complementation (BiFC) was employed in an attempt to detect SPS-SPP interactions visually. The findings clearly demonstrated that SPS interacts with SPP and that this interaction impacts soluble carbohydrate pools and affects carbon partitioning to starch. Moreover, a fusion construct between the two genes promotes plant growth in both transgenic Arabidopsis and hybrid poplar.