Phosphoinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. The dominant PI of the plasma membrane is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ₂ ]. One of only two PI(4,5)P ₂ recognition domains characterized in detail is the tubby domain. It is essential for targeting proteins into cilia involving reversible membrane association. However, the PI(4,5)P ₂ binding properties of tubby domains have remained enigmatic. Here, we used coarse-grained molecular dynamics simulations to explore PI(4,5)P ₂ binding by the prototypic tubby domain. The comparatively low PI(4,5)P ₂ affinity of the previously described canonical binding site is underpinned in a cooperative manner by a previously unknown, adjacent second binding site. Mutations in the previously unknown site impaired PI(4,5)P ₂ -dependent plasma membrane localization in living cells and PI(4,5)P ₂ interaction in silico, emphasizing its importance for PI(4,5)P ₂ affinity. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of tubby-like proteins that underlies delivery of ciliary cargo.