In the present work, we applied mechanical stimulus to study the propagations of intercellular calcium waves among BV-2 microglial cells by the fast fluorescence microscopy imaging system in vitro. The results showed that calcium signaling responded to mechanical stimulus and propagated to neighboring cells in isolated BV-2 microglial cells, suggesting that the paracrine pathway mediated intercellular calcium waves. Further data indicated that gap junction was involved in mechanically-induced intercellular calcium waves when the cells had physical contact with each other. More importantly, tunneling nanotubes (TNTs)-like structure between BV-2 microglial cells was found in vitro and mediated the propagation of intercellular calcium wave. Taken together, our studies demonstrated that three distinct pathways, including paracrine, gap junction and TNTs-like structures, could mediate the intercellular calcium waves in BV-2 microglial cells.