In recent years, the use of natural products for cancer prevention and treatment has received considerable attention. Bryostatin 1 is a natural macrocyclic lactone and a protein kinase D (PKD) modulator with potent antineoplastic properties that has been used to treat human cancers in clinical trials with limited success. Further understanding the mechanistic basis of Bryostatin 1 action may provide opportunities to improve clinical results of treatment with Bryostatin 1. We identified that PKD1, founding member of PKD family of serine/threonine kinases, modulates E-cadherin/beta-catenin activity, which plays an important role in cell integrity, polarity, growth, and morphogenesis. An aberrant expression and localization of E-cadherin/beta-catenin has been strongly associated with cancer progression and metastasis. In this study, we examined the effect of Bryostatin 1 treatment on PKD1 activation, beta-catenin translocation and transcription activity, and malignant phenotype of prostate cancer cells. Initial activation of PKD1 with Bryostatin 1 leads to colocalization of the cytoplasmic pool of beta-catenin with PKD1, trans-Golgi network markers, and proteins involved in vesicular trafficking. Activation of PKD1 by Bryostatin 1 decreases nuclear beta-catenin expression and beta-catenin/TCF transcription activity. Activation of PKD1 alters cellular aggregation and proliferation in prostate cancer cells associated with subcellular redistribution of E-cadherin and beta-catenin. For the first time, we have identified that Bryostatin 1 modulates beta-catenin signaling through PKD1, which identifies a novel mechanism to improve efficacy of Bryostatin 1 in clinical settings.