The HDAC-inhibitor sodium-butyrate, promotes differentiation of colon cancer cells as evidenced by induction of alkaline phosphatase (ALP) enzyme activity. Screening of a panel of 34 colon cancer cell lines identified cell lines responsive (44%) and refractory (56%) to butyrate-induced differentiation. Refractory cell lines were significantly enriched for those harbouring the CpG island methylator phenotype (CIMP)(P=0.016). The ALP gene family comprises 4 members - ALPi, ALPL, ALPP and ALPPL2 - each of which was induced by butyrate in responsive cell lines. ALPi promoter reporter studies identified a KLF/Sp1 cis element essential for butyrate-induction of ALPi promoter activity. Notably, butyrate-refractory cell lines displayed frequent methylation of a CpG dinucleotide located within this site in the endogenous ALPi promoter, as well as in KLF/Sp1 sites within the ALPL and ALPP promoters, indicating coordinate methylation of this gene family in select colon cancer cell lines. Notably butyrate-induction of an exogenous ALPi promoter-reporter paralleled induction of endogenous ALPi mRNA across the cell lines, suggesting the differential induction of ALPi may be due to selective induction of a common transcriptional regulator. To address this, responsive and refractory cell lines were treated with butyrate and transcription factors selectively induced in responsive lines identified by microarray. By focussing on factors which bind KLF/Sp1 regulatory elements, we identified KLF5 as selectively induced in responsive cell lines. Knockdown of KLF5 markedly attenuated butyrate-induction of ALP mRNA expression and enzyme activity. In summary, these findings demonstrate that the ALP gene family is co-ordinately inducible by butyrate in select colon cancer cell lines, in a KLF5-dependent manner. Cells lines refractory to butyrate-induction of the ALP gene family displayed coordinate promoter methylation of these genes. We propose that the coordinate methylation of this gene family in CIMP high colon cancers is likely linked to their coordinate transcriptional regulation.