Sweet potato [Ipomoea batatas(L.) Lam.] is an important food and industrial crop. Its storage root is rich in starch, which is present in the form of granules and represents the principal storage carbohydrate in plants. Starch content is an important trait of sweet potato controlling the quality and yield of industrial products. Vacuolar invertase encoding geneIbβfruct2was supposed to be a key regulator of starch content in sweet potato, but its function and regulation were unclear. In this study, threeIbβfruct2gene members were detected. Their promoters displayed differences in sequence, activity, andcis-regulatory elements and might interact with different transcription factors, indicating that the threeIbβfruct2family members are governed by different regulatory mechanisms at the transcription level. Among them, we found that onlyIbβfruct2-1show a high expression level and promoter activity, and encodes a protein with invertase activity, and the conserved domains and three conserved motifs NDPNG, RDP, and WEC are critical to this activity. Only two and six amino acid residue variations were detected in sequences of proteins encoded byIbβfruct2-2andIbβfruct2-3, respectively, compared withIbβfruct2-1; although not within key motifs, these variations affected protein structure and affinities for the catalytic substrate, resulting in functional deficiency and low activity. Heterologous expression ofIbβfruct2-1in Arabidopsis decreased starch content but increased glucose content in leaves, indicatingIbβfruct2-1was a negative regulator of starch content. These findings represent an important advance in understanding the regulatory and functional divergence among duplicated genes in sweet potato, and provide critical information for functional studies and utilization of these genes in genetic improvement.