Cancer stem cells (CSCs) contribute to the resistance of intractable prostate cancer, and dopamine receptor (DR)D2 antagonists exhibit anticancer activity against prostate cancer and CSCs. Human prostate cancer PC-3 cells were used to generate CSC-like cells, serving as a surrogate system to identify the specific DR subtype the inhibition of which significantly affects prostate-derived CSCs. Additionally, the present study aimed to determine the downstream signaling molecules of this DR subtype that exert more profound effects compared with other DR subtypes. The inhibitory effects of specific antagonists or small interfering (si)RNAs on DR subtypes were compared by analyzing morphological changes of cells, expression patterns of pluripotency markers, cell growth inhibitory activities and in vitro cell invasion. L-741,626, a specific DRD2 antagonist, induced morphological changes in PC-3-derived CSC-like cells, suppressed the expression of Oct4 (a pluripotency marker), and inhibited the growth of cells and tumors. The proliferation of heterozygous null PC-3 cells, generated using the CRISPR/Cas9 method, was slow, and their sphere-forming ability was substantially reduced, indicating a diminished capacity to produce CSCs. In addition, the phosphorylation of AMPK was suppressed by DRD2 siRNA and the heterozygous knockout of DRD2 in PC-3 cells, indicating that AMPK may be a putative downstream signaling molecule involved in the production and maintenance of PC-3-derived CSC-like cells. Specific inhibition or suppression of DRD2 caused PC-3-derived CSC-like cells to lose their properties and inhibited the formation of PC-3-derived CSC-like cells, followed by inhibition of the phosphorylation of AMPK, which is considered a putative downstream signaling molecule of DRD2. Further understanding of the mechanisms by which DRD2 regulates AMPK and the effects of AMPK inhibition on the properties of PC-3-derived CSC-like cells may provide valuable insights into the identification of molecular targets for treating intractable prostate cancer wherein AMPK is constitutively activated.