Abstract: Pre‐mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre‐mRNA splicing (rdm16‐4). The rdm16‐4 mutant displays a short‐root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16‐4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16‐4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full‐length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short‐root phenotype of rdm16‐4. This reveals that the RDM16‐mediated alternative splicing of cytokinin signaling components contributes to root growth.