In the brain, neurons communicate with each other at excitatory and inhibitory synapses. In each excitatory synapse, a dendritic spine receives the excitatory neurotransmitter released by an axon terminal. An extracellular high K⁺ induces robust depolarization and mimics massive synaptic transmission. A dendritic spine transiently shrinks in high K⁺ solution. In this study, we investigated the mechanisms of the spine shrinkage using cultured hippocampal neurons. The spine shrinkage observed in response to high K⁺ was inhibited in the presence of extracellular Co²⁺ or Cd²⁺. Then we investigated Ca²⁺-signaling pathway because these metal ions are supposed to be wide spectrum Ca²⁺-channel blockers. We preincubated neurons with ROCK inhibitor Y27632 or CaMKII inhibitor KN-93. However, neither of them inhibited the spine shrinkage. Finally, we tested whether myosin is involved in the spine shrinkage. A myosin ATPase inhibitor, blebbistatin, did not inhibit the spine shrinkage in high K⁺. Taken together, extracellular high K+-triggered Ca²⁺-influx seem to induce the spine shrinkage; however, the downstream signaling pathway or the motor machinery has not yet been determined.