© 2022 Kaan et al.Introduction. P-element induced wimpy testis-like 2 (Piwil2) is in the Piwi gene family. Piwil2 has important roles in the self-renewal mechanism of stem cell induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate, and cervical cancers. Glutathione S-transferase (GST) acts as detoxification in cancer metabolism. This study aimed to investigate the effects of the stem cell protein Piwil2 on MCF10A and MCF-7 at the GST activity levels. Materials/Methods. MCF-7/Piwil2 and MCF10A/Piwil2, transfected with a plasmid carrying the Piwil2 gene, and non-transfected MCF-7 and MCF10A were cultured in a complete DMEM/F12 medium. GST A1 and P1 activity was determined in these cell lines using as substrates CDNB, EA respectively. Results. According to experimental results, GST P1 activity decreased in the MCF-7/Piwil2 cells as compared with the non-transfected MCF-7 cells, however, MCF-7/Piwil2 cells demonstrated increases in GST A1 (total GST) activity. The statistically significant differences were found for the comparison of non-transfected MCF-7 and MCF-7/Piwil2 (p<0,0001), for GST enzyme activities by using CDNB and EA as substrates. These results were the same for the MCF10A cell line. Discussion. It is shown for the first time that transfection studies may affect GST activity at the cellular mechanism level. The study contributes to determining the effect of transfection on GST isoenzymes and also how the Piwil2 gene may affect GST activity in the stem cell line.