Hepatocyte nuclear factor 4α (HNF4α) is a dimeric transcription factor that controls as much as 60% of all liver genes. However, how it achieves such broad functional diversity is unknown. Here, we show that inflammation and immune pathway genes are differentially regulated in an isoform-dependent manner, confirming that each isoform homodimer preferentially regulates a subset of HNF4α targets. With all 12 human HNF4α isoform clones, we tested combinatorial pairings to determine whether isoform heterodimers are functional. Indeed, synergistic and potent pairing combinations of isoform heterodimers were noted for HNF4α3-8, HNF4α6-12, and HNF4α5-8 that activated CYP7A1, IL6, and IL17A genes, respectively. Surprisingly, these genes are not at all activated by their corresponding isoform homodimers, suggesting that a particular heterodimer pair can regulate its own subset of target genes. Given the combinatorial possibility of 66 isoform heterodimers, our data provide the basis for a more detailed understanding of the diverse influence of HNF4α.