Abstract Background There is evidence that calcium (Ca 2+ ) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha 1D -adrenergic receptor (alpha 1D -AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha 1D -AR to cross-talk with TRPV1 in PCa cell lines. Methods The expression of alpha 1D -AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha 1D -AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha 1D -AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca 2+ flux, IP 3 production, western blot and MTT assay. Results The levels of the alpha 1D -AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha 1D -AR and TRPV1 expression levels was found. Moreover, alpha 1D -AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha 1D -AR- and TRPV1-dependent protons release and Ca 2+ flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha 1D -AR/TRPV1 double knockdown. Conclusions We demonstrate a cross-talk between alpha 1D -AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha 1D -AR and TRPV1 channels.