PD0332991 (palbociclib/Ibrance®) is a cyclin-dependent kinase (CDK)4/6 inhibitor that has recently been approved for the treatment of estrogen receptor‑positive advanced breast cancer. The present study investigated the antiproliferative effects of PD0332991 on gastric cancer (GC) cells and the underlying molecular mechanisms. The activity of PD0332991 was tested in several GC cell lines, including AGS, KATO‑Ⅲ, NCI‑N87 and HS746T. Growth inhibitory activity of PD0332991, alone or in combination with fluorouracil (5‑FU), was measured by MTT assay. The effects of PD0332991 on cell cycle progression were analyzed by flow cytometry and western blotting. Protein pathway array and Ingenuity Pathway Analysis were used to identify signaling pathways that may mediate the antiproliferative effects of PD0332991. PD0332991 inhibited proliferation in a dose‑dependent manner and enhanced the activity of 5‑FU in all GC cell lines tested. Cells treated with PD0332991 exhibited cell cycle arrest in G1 phase of the cell cycle, whereas the number of cells in G2/M phase was decreased. PD0332991 also inhibited CDK6‑specific phosphorylation of retinoblastoma on Ser780, reduced the expression of cyclin D1, and induced expression of p53 and p27. Furthermore, 31 proteins were identified, the expression of which was significantly altered following treatment with PD0332991 in at least three cell lines. Pathway analysis indicated that the altered proteins were frequently associated with cell death, cell cycle and the molecular mechanism of cancer. The results of the present study indicated that PD0332991 may inhibit cell proliferation via modulation of the cell cycle, and may affect numerous oncogenic signaling pathways. Therefore, PD0332991 may be considered effective for the treatment of GC.