The sterol-independent regulatory element (SIRE) of the LDL receptor (LDLR) promoter mediates oncostatin M (OM)-induced transcription of the LDLR gene through a cholesterol-independent pathway. Our prior studies have detected specific associations of the zinc finger transcription factor Egr1 with the SIRE sequence in OM-stimulated HepG2 cells. Because the SIRE motif is composed of a c/EBP binding site and a cAMP response element, both of which are quite divergent from the classical GC-rich Egr1 recognition sequences, we hypothesized that Egr1 may regulate LDLR transcription through interacting with members of the c/EBP and CREB families. Here, we show that treating HepG2 cells with OM specifically leads to prominent increases of the levels of c/EBPbeta and Egr1 bound to the LDLR promoter in vivo. In vitro, the binding of Egr1 to the SIRE sequence is weak, but is strikingly enhanced in the presence of HepG2 nuclear extract. Mammalian two-hybrid assays demonstrate that the N-terminal transactivation domain of Egr1 specifically interacts with c/EBPbeta but not with c/EBPalpha or CREB. The OM treatment further enhances this interaction, resulting in a large increase in the Egr1 transactivating activity. The direct protein to protein contact between Egr1 and c/EBPbeta is also demonstrated by co-immunoprecipitation experiments. Furthermore, we show that a mutation of the phosphorylation motif of c/EBPbeta diminished the OM-stimulated interaction of Egr1 and c/EBPbeta. Taken together, we provide strong evidence that Egr1 regulates LDLR transcription via a novel mechanism of protein-protein interaction with c/EBPbeta.