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The ATP-binding cassette efflux transporter, ABCG2, is widely expressed in a variety of normal tissues, stem cells, as well as cancer cells. Existing data suggest that ABCG2 plays an important role in the maintenance of the stem cell phenotype and multidrug resistance of cancer cells. However, the potential role of ABCG2 in other cellular processes remains speculative and poorly understood. Here, we demonstrated that ABCG2 is involved in the proliferation of cancer cells. We used RNA interference approach to efficiently and specifically down-regulate ABCG2 protein levels in MCF-7/MX and A549 cells. We showed that knockdown of ABCG2 significantly inhibited the proliferation of these cells. Suppression of ABCG2 reduced the percentage of cells in the S phase of the cell cycle and enhanced G0/G1 accumulation. The G0/G1 growth arrest was associated with down-regulation of cyclin D3 and up-regulation of p21. Furthermore, blocking of ABCG2 function by chemical inhibitor fumitremorgin C also inhibited cell proliferation via the prolonged G0/G1 interval. Taken together, these findings suggest that ABCG2 correlates with cell cycle progression, highlighting a novel function of ABCG2 in cancer cell proliferation.