Abstract To date, there have been no tumor biomarkers validated and incorporated into oncologic practice for the early diagnosis of breast cancer. Recently, we showed that sperm-associated antigen 9 (SPAG9), a member of cancer testis (CT) antigen family, is associated with ovarian carcinomas. In the present study, we investigated SPAG9 expression and humoral immune response in breast cancer. We further evaluated the diagnostic potential of autoantibodies to SPAG9 protein in various stages, grades, and histotypes of breast cancer. We analyzed the association of SPAG9 immunoreactivity score (IRS) with predicted risk of breast cancer recurrence over 10 years. Our reverse transcription-PCR and immunohistochemical analyses revealed SPAG9 expression in 88% breast cancer specimens independent of tumor stages and grades. Further, the humoral immune response against SPAG9 was detected in 80% breast cancer patients with SPAG9-expressing tumors. The linear regression modeling predicted a direct relationship between presence of lymphovascular invasion and high SPAG9 IRS, whereas the univariate and multivariate logistic regression models predicted a strong association of SPAG9 IRS with tumor grade. Further, our data indicated a significant higher trend of SPAG9 IRS with the predicted high risk of breast cancer recurrence. The present investigation reports for the first time SPAG9 expression and humoral immune response in early stages and low-grade breast cancer. Although our data indicated that autoantibodies against SPAG9 represent a promising approach for the development of biomarker, further large-scale validation studies are required to establish its potential use in early diagnosis and monitoring of breast cancer recurrence. (Cancer Epidemiol Biomarkers Prev 2009;18(2):630–9)