Cell division is accompanied by dramatic changes in shape that ultimately lead to the physical separation of one cell into two. In 2D microenvironments, cells round up and remain adhered onto the substrate by thin retraction fibers during division. In contrast, in 3D environments, cells divide exhibiting long protrusions that guide the orientation of the division axis. However, the mechanism of cell division in three dimensions still remains poorly understood. Here we report the spontaneous formation of transient quasiperiodic membrane pearling on extended mitotic protrusions during 3D cell division. Protrusion membrane pearling may be initiated by the non-uniform distribution of focal adhesions and consequent stationary instability of the protrusive membrane. Overall, membrane pearling emergence may provide insights into a novel modality of 3D cell division with potential physiological relevance.