Background: ALDOA plays an essential role in cancer progression in different human cancers; however, its function has not been understood in prostate cancer (PCa). Methods: Associations of ALDOA expression with clinicopathological features and patient prognosis in PCa were evaluated based on data obtained from the Taylor database and our clinical tissue microarray. The potential roles of ALDOA in malignant progression were verified using a series of in vivo and in vitro experiments after stable ALDOA overexpression and knockdown in DU145 and PC3 cell lines. An aldolase A inhibitor was used to determine the effects of inhibition of ALDOA on PCa cell proliferation. Results: Higher expression of ALDOA was positively correlated with the incidence of postoperative metastasis and biochemical recurrence (BCR) and may predict poor prognosis in PCa patients. In vivo experiments demonstrated that overexpression of ALDOA could significantly promote cell proliferation, prolong the cell cycle, and significantly reduce the apoptosis rate of PCa cells. Knockdown of expression of ALDOA could inhibit the proliferation and shorten the cell cycle of PCa cells significantly, with no significant effects on cell apoptosis (P > 0.05). In vitro experiments showed that overexpression of ALDOA could significantly promote tumor growth (P < 0.05), while treatment with the Aldolase A inhibitor naphthol AS-E phosphate dose-dependently suppressed the growth of PCa cells (P < 0.01). The analysis of datasets from the Taylor database showed that there was negative regulatory relationship between the expression of ALDOA and MYPT1 (P < 0.001). Conclusion:Our study revealed that ALDOA played an important role in the progression of PCa. The MYPT1-ALDOA signaling axis may be a new target for the clinical treatment of PCa patients given its negative regulatory relationship. Our study suggests that Aldolase A inhibitors may represent a novel approach to inhibit the growth of PCa.