Vitamin D receptor (VDR) activation is associated with cardiovascular benefits in chronic kidney disease patients, but whether VDR's hormone and prehormone exhibit similar effects requires more studies. Neonatal rat cardiomyocytes were treated with VDR agonists (calcitriol and/or paricalcitol) and the prehormone calcidiol in the presence of aldo (1μM). The expression of VDR target genes were determined by real-time PCR and Western blotting. The expression and activity of CYP27B1 (the enzyme responsible for converting calcidiol to calcitriol) was measured. Treating cells with aldo (1μM) for 24h significantly reduced the VDR mRNA (29%) and protein levels (>90%). Calcitriol and calcidiol induced VDR expression in the presence of aldo with EC(50) at 0.3 and 7,952nM, respectively. Calcitriol, paricalcitol and calcidiol stimulated CYP24A1 (EC(50) at 6.4, 4.5 and 992nM, respectively) and suppressed NPPB expression (IC(50) at 1.9, 0.1 and 210nM, respectively) in the presence of 1μM aldo. Neonatal rat cardiomyocytes expressed CYP27B1 and converted calcidiol to calcitriol at a low rate (~10% in 24h). VDR hormone calcitriol and its analog paricalcitol exhibit more potent effects than the prehormone calcidiol in cardiomyocytes.