Mutations of the nuclear lamins cause a wide range of human diseases, including Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome. Defects in A-type lamins reduce nuclear structural integrity and affect transcriptional regulation, but few data exist on the biological role of B-type lamins. To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts from Lmnb1(Delta/Delta) and wild-type littermate embryos by time-lapse videomicroscopy. Here, we report that Lmnb1(Delta/Delta) cells displayed striking nuclear rotation, with approximately 90% of Lmnb1(Delta/Delta) nuclei rotating at least 90 degrees during an 8-h period. The rotation involved the nuclear interior as well as the nuclear envelope. The rotation of nuclei required an intact cytoskeletal network and was eliminated by expressing lamin B1 in cells. Nuclear rotation could also be abolished by expressing larger nesprin isoforms with long spectrin repeats. These findings demonstrate that lamin B1 serves a fundamental role within the nuclear envelope: anchoring the nucleus to the cytoskeleton.