Panaxydol, a polyacetylenic compound derived from Panax ginseng has been reported to suppress the growth of cancer cells. However, the molecular mechanisms underlying cell cycle arrest by this compound in non-small cell lung cancer (NSCLC) are unknown. Our study found that panaxydol treatment induced cell cycle arrest at G₁ phase in NSCLC cells. The cell cycle arrest was accompanied by down-regulation of the protein expression of cyclin-dependent kinase (CDK) 2, CDK4, CDK6, cyclin D₁ and cyclin E, and decrease in the phosphorylation of retinoblastoma (Rb) protein. Furthermore, up-regulation of cyclin-dependent kinase inhibitor (CDKI) p21^CIP1/WAF1 and p27^KIP1 was observed in panaxydol-treated NSCLC cells. In addition, panaxydol also induced accumulation of intracellular Ca²⁺ ([Ca²⁺]_i). (Acetyloxy)methyl 2-({2-[(acetyloxy)methoxy]-2-oxoethyl}[2-(2-{2-[bis({2-[(acetyloxy)methoxy]-2-oxoethyl})amino]phenoxy}ethoxy)phenyl]amino)acetate (BAPTA-AM), the Ca²⁺ chelator, attenuated not only panaxydol-induced accumulation of [Ca²⁺]_i, but also G₁ cell cycle arrest and decrease of CDK6 and cyclin D₁ protein expression level. These results demonstrated that the anti-proliferative effects of panaxydol were caused by cell cycle arrest, which is closely linked to the up-regulation of [Ca²⁺]_i and represents a promising approach for the treatment of lung cancer.