In yeast, osmotic upshock causes a release of vacuolar Ca²⁺ through the mechanosensitive transient receptor potential channel, Yvc1. We screened the collection of 4810 yeast gene deletants twice for alterations in this response in an attempt to find elements that regulate the amount of vacuolar Ca²⁺ or the Yvc1 channel. Severe overresponders and underresponders to upshock were further scrutinized for their calcium content with ⁴⁵Ca and their Yvc1 electrophysiological activities under patch-clamp. The severe underresponders have lower calcium content but no change in Yvc1 activity. The strong overresponders, most of which are deleted of genes involved in cell wall metabolism, have higher calcium content. Wall mutations are known to up-regulate Ca²⁺-calcineurin-dependent genes. It appears that stress on the cell wall induces Ca²⁺ accumulation, adaptively anticipating the need in defense or repair against future stress, including osmotic stress.—Loukin, S., Zhou, X., Kung, C., Saimi, Y. A genome-wide survey suggests an osmoprotective role for vacuolar Ca²⁺ release in cell wall-compromised yeast.