The purpose of this study was to clarify the role of human beta defensin-1 (hBD-1) in predicting PAC in morphologically normal prostate glands. In total, 25 patients with a negative initial biopsy for PAC and diagnosed as PAC positive in subsequent biopsies performed within 1 year of the initial biopsy were included. As a control group, 22 patients negative for PAC in at least three consecutive histologic examinations were selected. Expression of hBD-1 was analyzed separately via immunohistochemistry in paired cores of non-neoplastic gland and PAC in the false-negative group and control group. Loss of hBD-1 expression was observed in 95.6% and 90.0% PAC cases with Gleason Patterns 3 and 4 in repeat biopsies, respectively. hBD-1 loss of basal cells in 40 (85.1%) previous non-neoplastic biopsy cores in the false-negative group was observed, in contrast to preserved basal cell expression of hBD-1 in 64 (72.7%) biopsy cores in the control group (p = 0.001). Multivariate logistic regression analysis showed that hBD-1 basal cell loss (≥20% of prostatic glands in total cores) is an independent factor for predicting PAC (odds ratio: 4.739, confidence interval: 1.093-20.554, p = 0.038). hBD-1 loss of basal cells is a useful indicator to identify extremely high-risk patients with initially negative biopsy.