A polymorphism at codon 655 (ATC/isoleucine to GTC/valine [Ile655Val], rs1801200) in the transmembrane domain-coding region of human ERBB2 gene has been previously evaluated for its association with breast cancer risk with mixed results. We evaluated this polymorphism in association with breast cancer in a group of women who participated in a large-scale, population-based, case-control study of breast cancer in Shanghai, China, followed by an in vitro analysis of the function of this polymorphism. Genomic DNA from 3,012 patients with breast cancer and 3,004 healthy controls was examined for the Ile655Val polymorphism using a TaqMan genotyping method. Adjusted odds ratios (OR) were derived from multiple logistic regression. In vitro analyses were carried out to examine whether the Ile655Val polymorphism affect ERBB2 expression and the activity of its downstream targets. Approximately 2% of study subjects carry the Val/Val genotype. Compared with women with the Ile/Ile (76%) genotype, women who had the Ile/Val (22%) or Val/Val genotype did not have an elevated risk of breast cancer. Stratified analyses by age and menopausal status revealed no apparent association with this polymorphism in any subgroups of women. In a serious of biochemical analyses, we found that the Ile655Val substitution did not alter ErbB2 and its downstream signaling molecule activity. These study results suggest that Ile655Val polymorphism of the ERBB2 gene do not alter its activity and may not be associated with increased breast cancer risk among Chinese women.