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Nowadays, most studies focused on cancer stem cells (CSCs) through their abilities to cause tumorigenicity, drug resistance, and cancer recurrence. On the other side, nonsteroidal anti‐inflammatory drugs (NSAIDs) have been taken into consideration because of cheapness and availability. For the reasons mentioned above, we have studied the effect of ibuprofen as an NSAID on CSCs derived from AGS and MKN‐45 gastric cancer cell lines to perform effective cancer therapy. We evaluated cell viability, spheroid body formation, monolayer, and soft agar colony formation to express the anti‐cancer effect of ibuprofen on CSCs. Also, real‐time RT‐PCR data of stemness markers and genes affected on, or downstream of Wnt signaling pathway were evaluated. Our findings suggest that ibuprofen at 1,000 μM for 48 h can reduce cell proliferation, stemness features in CSCs by changing the expression level of CD44, OCT3/4, SOX2, Nanog, and KLF4 as stemness markers. Furthermore, ibuprofen can have an inhibitory role in Wnt signaling pathway by changing the expression level of some genes, including CTNNB1, CTNNBIP1, SMARCD1, PYGO2, SUFU, CASK, and KREMEN1. According to our study, ibuprofen has an anti‐proliferative effect on CSCs derived from AGS and MKN‐45 cells.