Background: A computer-aided diagnosis system for bone scan with semiquantitative index of Bone Scan Index (BSI) may be used to quantify the spread of bone metastases. This retrospective study aimed to examine associations between BSI, bone metabolic markers, and prostate specific antigen (PSA) in prostate cancer patients with bone metastases. Methods: A total of 158 scintigraphy of 52 patients (mean examinations/person 3, range 1-8; mean age 71 years, range 46 to 86) were studied. The intervals between bone scan and blood examinations were 0 to 16 days (median 0 day). Subjects were divided into 4 groups according to BSI; Group A: 0 - <2, Group B: 2 - <4, Group C: 4 - <8, and Group D: 8<. The markers of PSA, pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (1-CTP), bone alkaline phosphatase (BAP), and tartate resistant acid phosphatase-5b (TRAP-5b) were examined. As the values of PSA, BAP, and TRAP-5b covered a large range of scales, we also used the logarithms of the variables. BSI, which corresponded amount of metastatic lesions, was automatically calculated by BONENAVI software (FUJIFILM RIPharma, Co. Ltd., Tokyo, Japan; Exini Bone, Exini Diagnostics, Sweden). Results: All scans showed increased uptake at bone metastases. BSI correlated significantly with 1-CTP, BAP, logBAP, TRAP-5b, logTRAP-5b, and logPSA (r=0.39, 0.66, 0.71, 0.69, 0.61 and 0.41, respectively). Statistical F value was 11 in 1-CTP, 31 in BAP, 29 in logBAP, 19 in TRAP-5b, 14 in logTRAP-5b, 3 in PSA, and 9 in logPSA by ANOVA, respectively. Comparison by Dunnett’s test showed significantly higher values in Group D for all bone metabolic markers and logPSA, Group C for BAP, logBAP, TRAP-5b, and logTRAP-5b, and Group B for logTRAP-5b compared with Group A, respectively. However, PSA did not correlate significantly with extent of bone metastases as assessed by BSI. Conclusions: The changes in BSI showed close relationship with all bone metabolic markers but not with PSA. The BSI reflected the activity and extent of bone metastases, and might be used as an imaging biomarker.