This study delves into the role of SF3B3, a component of U2 small nuclear ribonucleoproteins, in colorectal cancer (CRC). Researchers found SF3B3 to be upregulated in CRC samples and associated with poor survival. Inhibiting SF3B3 suppressed CRC cell proliferation and metastasis by inducing mitochondria injury, increasing reactive oxygen species (ROS), and promoting apoptosis. Mechanistically, SF3B3 silencing altered mTOR exon-skipping splicing, suppressing lipogenesis through mTOR-SREBF1-FASN signaling. Combining SF3B3 inhibition with mTOR inhibitors showed synergistic antitumor effects in CRC organoids and xenografts. Additionally, SF3B3 was identified as a crucial regulator of mTOR splicing and autophagy in various cancers, underscoring its potential as a therapeutic target.