We examined the role of the extracellular signal regulated kinases (ERK) in 1,25‐dihydroxyvitamin D (1,25(OH)₂D₃)‐induced gene expression in the differentiated Caco‐2 cells. 1,25(OH)₂D₃‐regulated expression of the 25‐hydroxyvitamin D, 24‐hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH)₂D₃‐regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5‐mediated phosphorylation of the transcription factor Ets‐1 enhanced 1,25(OH)₂D₃‐mediated CYP24 gene transcription in proliferating but not differentiated Caco‐2 cells due to reduced levels of ERK5 and Ets‐1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH)₂D₃‐induced 3X‐VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co‐activator recruitment in ERK‐modulation of vitamin D‐regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH)₂D₃ treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH)₂D₃‐regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH)₂D₃ effects on the CYP24 promoter by MED1‐mediated events. Our data show that there are both promoter‐specific and cell stage‐specific roles for the ERK signaling pathway on 1,25(OH)₂D₃‐mediated gene induction in enterocyte‐like Caco‐2 cells. J. Cell. Physiol. 219: 132–142, 2009. © 2008 Wiley‐Liss, Inc.