Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in the transcriptional regulation of cardiomyocyte hypertrophy, through the phosphorylation of class II histone deacetylase (HDAC) enzymes, such as HDAC5. To identify novel molecular partners for PKD1 in the heart, we performed a yeast two-hybrid screen of a human cardiac cDNA library, which revealed FHL2 as an interaction partner for the PKD1 catalytic domain. Pull-down assays using recombinant GST fusion proteins confirmed the interaction of heterologously expressed PKD1 in adult rat ventricular myocytes (ARVM) with not only FHL2 but also FHL1, another FHL family member. The ability of endogenously expressed PKD1 to also associate with FHL1 and FHL2 was confirmed by performing GST pull-down assays in neonatal ventricular myocytes (NRVM), in which PKD1 is more abundant than in ARVM. In both ARVM and NRVM, the FHL1/2-PKD1 interaction was independent of PKD1 activation status. To investigate the FHL1/2-PKD1 interaction in the cellular environment, a GFP-PKD1 fusion protein was expressed in NRVM by adenoviral gene transfer and the associated protein complex was extracted using GFP-Trap® methodology. Subsequent immunoblot analysis revealed that both FHL1 and FHL2 associate with GFP-PKD1 (but not GFP alone, expressed in controls). In vitro kinase assays using recombinant FHL1 or FHL2 showed that neither protein is a PKD1 substrate. To explore the potential functional role of the FHL1/2-PKD1 interaction, the expression of FHL1, FHL2 or both was silenced in NRVM by siRNA transfection. Selective knock-down of FHL1 expression (by ∼75%) significantly inhibited PKD1 activation (as reflected by PKD1 autophosphorylation) and HDAC5 phosphorylation in response to endothelin-1 (ET-1), but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knock-down of FHL2 expression (by ∼60%) caused a significant reduction in PKD1 activation and HDAC5 phosphorylation in response to both stimuli. Simultaneous down-regulation of FHL1 and FHL2 expression had the same impact on ET-1-induced PKD1 activation and HDAC5 phosphorylation as the selective silencing of either FHL gene alone, indicating that FHL1 and FHL2 proteins are both necessary components of the same signalling module downstream of the ET-1 receptors. We conclude that FHL1 and FHL2 are novel cardiac interaction partners for PKD1 that differentially facilitate PKD1 activation and HDAC5 phosphorylation by distinct neurohormonal stimuli that can trigger transcriptional reprogramming towards cardiomyocyte hypertrophy.