An increase in intrapulpal hydrostatic pressure (HP) occurs during inflammation and restorative procedures. However, the role that HP plays in human dental pulp cell (HDPCs) is not well understood. In this study, we investigated the effect of HP on interleukin-6 (IL-6) expression in HDPCs. The results showed that HP up-regulated IL-6 HDPC mRNA expression and protein release in a time- and dose-dependent manner. The induction of IL-6 by HP was significantly inhibited by an antagonist for the non-specific purinergic receptor family (suramin) and an intracellular calcium chelator (BAPTA-AM). These results suggest the involvement of P2Y receptor and intracellular calcium in HP signaling pathway, respectively. Using loss of function experiments, we showed MRS2578 (a specific P2Y6 antagonist), as well as P2Y6 small interfering RNA, abolished HP-induced IL-6, while MRS2179 (a specific P2Y1 antagonist) and NF449 (a P2X1, P2X3, P2Y1, and P2Y2 antagonist) had no effect. Moreover, we demonstrated that either the conditioned medium collected from the culture after application of HP or addition of UDP, a selective agonist of P2Y6, up-regulated IL-6 expression in HDPCs. In conclusion, this study is the first demonstration that HP could induce IL-6 expression through the P2Y6 receptor in HDPCs, which provides new insight into the role of pressure on cytokine release during pulpal inflammatory process.