The activation of hepatic stellate cells (HSCs) is an essential part in the development of alcoholic liver fibrosis (ALF). In this study, stimulated HSCs with 200 μM acetaldehyde for 48 h was used to imitate alcoholic liver fibrosis in vitro. The western blot and qRT-PCR results showed that P2X7R expression was significantly increased in the activation of HSCs after acetaldehyde treatment. Interestingly, activation of P2X7R by stimulating with P2X7R agonist BzATP significantly promoted acetaldehyde-induced CyclinD1 expression, cell proportion in S phase, inflammatory response, and the protein and mRNA levels of α-SMA, collagen I. In contrast, blockage of P2X7R by stimulating with the inhibitor A438079 or transfecting with specific siRNA dramatically suppressed acetaldehyde-induced HSCs activation. Furthermore, PKC activation treated with PMA could obviously up-regulate the expression of α-SMA and collagen I and the phosphorylation of GSK3β, while inhibition of PKC significantly reduced GSK3β activation. Moreover, GSK3β inhibition harvested a dramatic decrease of the mRNA and protein levels of α-SMA and collagen I by suppressing GSK3β phosphorylation. Taken together, these results suggested that purinergic P2X7R mediated acetaldehyde-induced activation of HSCs via PKC-dependent GSK3β pathway, which maybe a novel target for limiting HSCs activation.