Insulin-like growth factor-1 (IGF-1) is an important growth factor for breast cancer cells and insulin-like growth factor binding protein-3 (IGFBP-3) its most prevalent binding protein. Prostate-specific antigen (PSA) enzymatically cleaves IGFBP-3 into fragments (BP3-FR). Our purpose was to determine the association of these markers in nipple aspirate fluid (NAF) and serum with the presence of breast cancer. NAF from 175 and serum from 215 subjects were collected from women with or without breast cancer. In unadjusted analysis low NAFPSA (P < 0.001) and high NAFIGFBP-3 (P = 0.023) were associated with breast cancer. Low serum PSA was associated with postmenopausal breast cancer (P = 0.034). In separate multivariate analyses, controlling for age, menopausal status, and age at menarche, NAF PSA and IGFBP-3 were each associated with breast cancer. The association was significant for NAF IGFBP-3 in all women (P = 0.031), but for NAF PSA only in premenopausal women (P < 0.001). When considered jointly, only NAF PSA was significant. Therefore, NAF PSA, and to a lesser extent NAF IGFBP-3 and serum PSA, seem to be important predictors of breast cancer.