The phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway is one of the most commonly mutated pathways in breast cancer. To date, there has been no study on polymorphism of phosphatidylinositol-3,4-bisphosphonate 3-kinase, catalytic subunit alpha (PIK3CA) gene microsatellites and their link with breast cancer risk. In the present study, we investigate the guanine-thymine (GT) dinucleotide repeat polymorphism in intron 1 of PIK3CA gene in a cohort of 200 breast cancer patients and 200 healthy individuals and its link to the risk of developing breast cancer. The results of this study demonstrate that PIK3CA gene allele distribution in Isfahan population varies between 13 and 20 repeats. GT14 and GT16 were the most common allele present in patients, and GT17 was the most common allele in controls. Women with one or two alleles shorter than 17 GT repeat have a significantly higher risk of developing breast cancer [Odds ratio (OR) 3.6, p = 0.00001 and OR 3.98, p = 0.000001, respectively], in contrast, women with one or two alleles longer than 16 are at lower risk of breast cancer. This result suggests a potential role for this microsatellite as a predictive marker of breast cancer risk in Iranian women.