Compared to prostate adenocarcinoma, neuroendocrine prostate carcinoma is characterized by aggressive tumor biology and a poor prognosis. Several studies have focused on tumor markers to assess neuroendocrine differentiation and treatment outcomes. However, few studies have investigated changes in tumor markers throughout the clinical course. This observational study evaluated neuroendocrine differentiation, survival outcomes, and temporal changes in neuroendocrine marker positivity in patients with prostate cancer. A total of 193 prostate cancer cases were included in this study. We measured circulating neuroendocrine tumor markers, including neuron-specific enolase (NSE) and pro-gastrin-releasing peptide (pro-GRP), and calculated their positivity rates. In relation to castration-resistant prostate cancer (CRPC), we assessed the changes in the positive rates of neuroendocrine tumor markers over time. In the cross-sectional cohort, the positivity rate of circulating neuroendocrine markers was 19.8% in hormone-sensitive prostate cancer and 64.9% in CRPC (24/37). Temporal changes in marker positivity were evaluated in a separate longitudinal CRPC follow-up cohort (n = 39) with serial NSE/pro-GRP measurements. In the context of CRPC, pro-GRP was significantly associated with prognosis. The median overall survival for patients with normal pro-GRP levels was 78 months, whereas that for patients with elevated pro-GRP levels was significantly shorter (36.5 months, p = 0.0216). In the longitudinal CRPC follow-up cohort (n = 39), the overall marker positivity rate increased from 61.5% at baseline to 92.3% at approximately four years. Neuroendocrine tumor markers were significantly elevated in CRPC and correlated with survival outcomes. The positivity rate of these tumor markers increased during CRPC treatment. This suggests that treatment-emergent neuroendocrine differentiation may occur more frequently than previously anticipated.