The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) inhibits the growth of prostate epithelial cells, however the underlying mechanisms have not been clearly delineated. In the current study, the impact of 1,25(OH)2D on the rapid activation of extracellular-regulated kinase (ERK) 1/2 and protein kinase C α (PKCα), and the role of these pathways in growth inhibition was examined in immortalized mouse prostate epithelial cells, MPEC3, that exhibit stem/progenitor cell characteristics. 1,25(OH)2D treatment suppressed the growth of MPEC3 in a dose and time dependent manner (e.g., 21% reduction at three days with 100 nM 1,25(OH)2D treatment). However, ERK1/2 activity was not altered by 100 nM 1,25(OH)2D treatment for time points from 1 min to 1 h in either serum-containing or serum-free medium. Similarly, PKCα activation (translocation onto the plasma membrane) was not regulated by short-term treatment of 100 nM 1,25(OH)2D. In conclusion, 1,25(OH)2D did not mediate rapid activation of ERK1/2 or PKCα in MPEC3 and therefore the growth inhibitory effect of 1,25(OH)2D is independent of rapid activation of these signaling pathways in this cell type.