There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.