Osteopontin (OPN) is a sialic acid-rich phospoprotein secreted by a wide variety of cancers. We have previously shown that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. While a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC colonic epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nt-107 to nt-174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared to YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt-101 to nt-123 and nt-121 to nt-145, which specifically bind CT26 nuclear proteins. Competition with unlabelled mutant oligonucleotides revealed that the regions, nt-115 to -118 and nt-129 to -134, were essential for protein binding. Subsequent supershift and ChIP assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein.