Centrosomes nucleate and organize interphase MTs and are instrumental in the assembly of the mitotic bipolar spindle. Here we report that two members of the multifunctional protein 4.1 family have distinct distributions at centrosomes. Protein 4.1R localizes to mature centrioles whereas 4.1G is a component of the pericentriolar matrix surrounding centrioles. To selectively probe 4.1R function, we used RNA interference-mediated depletion of 4.1R without decreasing 4.1G expression. 4.1R downregulation reduces MT anchoring and organization at interphase and impairs centrosome separation during prometaphase. Metaphase chromosomes fail to properly condense/align and spindle organization is aberrant. Notably 4.1R depletion causes mislocalization of its binding partner NuMA (Nuclear Mitotic Apparatus Protein), essential for spindle pole focusing, and disrupts ninein. During anaphase/telophase, 4.1R-depleted cells have lagging chromosomes and aberrant MT bridges. Our data provide functional evidence that 4.1R makes crucial contributions to centrosome integrity and to mitotic spindle structure enabling mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.