Alternative splicing can produce transcripts that encode proteins with altered functions. The transcripts of the ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) gene, which is an important component in abscisic acid (ABA) signaling, are subjected to alternative splicing in both monocotyledons and dicotyledons. We identified two alternatively spliced tomato (Solanum lycopersicum) SlABI3 transcripts, SlABI3-F and SlABI3-T, which encode the nucleus-localized full-length and truncated proteins, respectively. The tissue-specific accumulation of SlABI3-F and SlABI3-T was determined, particularly in seeds at different developmental stages and in response to phytohormonal and abiotic stress. Ectopic over-expression of SlABI3-F and SlABI3-T resulted in the induction of seed-specific genes SlSOM, SlEM1 and SlEM6 in vegetative tissues. However, over-expression of SlABI3-F, but not SlABI3-T, activated expression of the downstream gene SlABI5 and conferred hypersensitivity to exogenous ABA during seed germination and primary root growth. In addition, the SlABI3-F protein interacted with SlABI5 much stronger than SlABI3-T did in the yeast two-hybrid assay. These results suggest that SlABI3-F and SlABI3-T have similar and distinct functionality in the ABA signaling, dependent on which tissue/organ they accumulate in.