Heme is an essential requirement for cell survival. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and consists of two isozymes, HO-1 and HO-2. To identify the protein that regulates the expression or function of HO-1 or HO-2, we searched for proteins that interact with both isozymes, using protein microarrays. We thus identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) that synthesizes or degrades fructose-2,6-bisphosphate, a key activator of glycolysis, depending on cellular microenvironments. Importantly, HO-2 and PFKFB4 are predominantly expressed in haploid spermatids. Here, we show a drastic reduction in expression levels of PFKFB4 mRNA and protein and HO-2 mRNA in HepG2 human hepatoma cells in responses to glucose deprivation (≤ 2.5 mM), which occurred concurrently with remarkable induction of HO-1 mRNA and protein. Knockdown of HO-2 expression in HepG2 cells, using small interfering RNA, caused PFKFB4 mRNA levels to decrease with a concurrent increase in HO-1 expression. Thus, in HepG2 cells, HO-1 expression was increased, when expression levels of HO-2 and PFKFB4 mRNAs were decreased. Conversely, overexpression of HO-2 in HepG2 cells caused the level of co-expressed PFKFB4 protein to increase. These results suggest a potential regulatory role for HO-2 in ensuring PFKFB4 expression. Moreover, in D407 human retinal pigment epithelial cells, glucose deprivation decreased the expression levels of PFKFB4, HO-1, and HO-2 mRNAs. Thus, glucose deprivation consistently down-regulated the expression of PFKFB4 and HO-2 mRNAs in both HepG2 cells and RPE cells. We therefore postulate that PFKFB4 and HO-2 are expressed in a coordinated manner to maintain glucose homeostasis.