Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations inSF3B1are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenicSF3B1WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3′ splice sites uponSF3B1mutation, including an event in theMAP3K7gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and inSF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towardsSF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.