This study aimed to elucidate the functional correlation between G-protein activation and Ca²⁺ signaling during hypotonic shock-induced cell swelling in human osteoblast-like cells. Calcium influx was investigated using fura-2 fluorescence imaging, specific channel blockers, and G-protein modulators such as cholera toxin (CTX) and pertussis toxin (PTX). The external solution contained hypotonic Na⁺, and 300 M Cd²⁺ was applied to assess the involvement of extracellular Ca²⁺ channels. Calcium influx was significantly inhibited by Cd²⁺, indicating an extracellular source of Ca²⁺ through specific channels. CTX prolonged the hypotonic Na⁺-induced elevation of intracellular Ca²⁺, while PTX completely abolished it. These results suggest the involvement of G-protein-mediated pathways in Ca²⁺ regulation. Furthermore, the CTX-induced potentiation implies regulation via the cAMP/protein kinase A signaling axis, indicating that stretch sensitivity may be modulated by hormone-activated adenylate cyclase. This study is the first to demonstrate a functional link between hypotonic stress-induced Ca²⁺ influx and G-protein activation in human osteoblast-like cells, potentially involving a novel Ca²⁺ channel regulated by the cAMP/PKA pathway.