Homeobox B13 (HOXB13) and trefoil factor 3 (TFF3) are novel candidates for the classification of prostate cancer (PC) in molecular subtypes that could predict the clinical evolution of patients. The aim of our study was to analyze the possible associations between HOXB13 and TFF3 immunohistochemical (IHC) expression in sporadic prostate adenocarcinoma (PAC), the potential prognostic value in relation to the classical clinico-pathological parameters, as well as their role in defining distinct molecular subtypes of this malignancy. The study group comprised 105 patients diagnosed with PAC who underwent radical prostatectomy. IHC exam was performed using anti-HOXB13 and anti-TFF3 antibodies and a scoring system that permit the separation of the cases into two subgroups, with low and high immunoexpression, respectively. The statistical analysis evaluated the relationship between the two immunomarkers and clinico-pathological parameters. The Kaplan-Meier curves and log-rank Mantel-Cox test were used for assessing the prostate-specific antigen (PSA)-progression free survival. Four subgroups of PAC were defined based on the IHC overexpression and low immunoexpression of HOXB13 and TFF3. High HOXB13 and TFF3 immunoexpression was commonly identified in cases characterized by a Gleason score over 7, a G4 or G5 dominant pattern, a grade group of 3 or 4 and a preoperatory PSA serum level over 20 ng/mL. HOXB13 overexpression was also associated with pathological tumor-node-metastasis (pTNM) stage. The subgroup with both low HOXB13 and TFF3 immunoexpression had the highest PSA-progression free interval, whereas the subgroup with high HOXB13 immunoexpression and low TFF3 immunoexpression presented the lowest rate, but no statistically significant differences were registered. Our results sustain the role of HOXB13 and TFF3 in the stratification of PAC. Further investigations in larger cohorts are imposed to validate the clinical significance of these subgroups in the diagnostic and prognostic of PAC.