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Objective To investigate whether prostate-specific antigen (PSA) fluctuation correlates with a prostate cancer and to assess whether PSA fluctuation could be used for diagnosis of prostate cancer. Materials and Methods Our study included 229 patients who were performed a prostate biopsy (non-cancer group, 177; prostate cancer group, 52). Enrolled patients were provided twice PSA tests within 6 months. PSA fluctuation (%/month) was defined as a change rate of PSA per a month. Independent t test was used to compare between two groups. Receiver operator characteristic curve was used to assess the availability as a differential diagnostic tool and the correlation. Simple linear regression was performed to analyze a correlation between PSA fluctuation and other factors such as age, PSA, PSA density, and prostate volume. Results There were significant differences in PSA, PSA density, percentage of free PSA, and PSA fluctuation between two groups. PSA fluctuation was significantly greater in non-cancer group than prostate cancer group (19.95±23.34%/month vs 9.63±8.57%/month, P=0.004). The most optimal cut-off value of PSA fluctuation was defined as 8.48%/month (sensitivity, 61.6%; specificity, 59.6%; AUC, 0.633; P=0.004). In a simple linear regression model, only PSA level was significantly correlated with PSA fluctuation. Conclusion Patients with wide PSA fluctuations, although baseline PSA levels are high, might have a low risk of diagnosis with prostate cancer. Thus, serial PSA measurements could be an option in patients with an elevated PSA level.