Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in a great structural and molecular diversity of dendritic spines. Active spines with large Ca2+-transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines by the actin-based motor myosin V. An increase in synaptic strength often correlates with stable anchoring of the ER, followed by the formation of the spine apparatus organelle. Here we characterize the newly identified interaction of myosin V with the Ca2+-sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. We propose that caldendrin transforms myosin into a stationary F actin tether, and show that in mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway.