Abiotic stresses profoundly affect plant growth and development and limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich splicing factors play a key role in pre-mRNA splicing to regulate different biological processes under stress conditions. Alternative splicing (AS) of SR transcripts and other transcripts of stress-responsive genes generates multiple splice isoforms that contribute to protein diversity, modulate gene expression, and affect plant stress tolerance. Here, we investigated the function of the plant-specific SR protein RS33 in regulating pre-mRNA splicing and abiotic stress responses in rice. The loss-of-function mutant rs33 showed increased sensitivity to salt and low-temperature stresses. Genome-wide analyses of gene expression and splicing in wild-type and rs33 seedlings subjected to these stresses identified multiple splice isoforms of stress-responsive genes whose AS are regulated by RS33. The number of RS33-regulated genes was much higher under low-temperature stress than under salt stress. Our results suggest that the plant-specific splicing factor RS33 plays a crucial role during plant responses to abiotic stresses.