Flower development is of great significance for plant reproductive growth, but the molecular mechanisms underlying flower development remain to be fully understood. In this study, a tomato (Solanum lycopersicum L.) Divergent C1 (DC1) domain protein SlCHP16 was identified as a negative regulator of flower development. Overexpression of SlCHP16 led to the delay of flower bud development and failure of flowers to blossom and bear fruits. Conversely, down-regulation of SlCHP16 transcripts, via RNA interference (RNAi), led to formation of larger flowers in transgenic tomato plants. In SlCHP16-overexpressing plants, floral primordia and floral organs were initiated normally, but their subsequent growth and development were severely arrested. Transcriptome analysis showed that this arrest was associated with the changes in expression levels of a large number of genes involved in cell division and organ development. Tomato 14-3-3 protein 12 (TFT12) was identified as an interacting protein of SlCHP16 by tandem mass spectrometry, and its overexpression in tomato plants led to the formation of enlarged flowers. The presence of SlCHP16 disturbed the stability and homodimerization of TFT12 in plant cells. The results of this study demonstrate an inhibitory role of SlCHP16 in flower development in tomato by interaction with the 14-3-3 protein TFT12. This work provides new insights into the mechanisms that control development of floral organs.