Farnesoid X receptor α (FXRα, NR1H4) is a bile acid-activated nuclear receptor that regulates the expression of glycolytic and lipogenic target genes by interacting with the 9-cis-retinoic acid receptor α (RXRα, NR2B1). Along with cofactors, the FXRα proteins reported thus far in humans and rodents have been observed to regulate both isoform (α1-4)- and tissue-specific gene expression profiles to integrate energy balance and metabolism. Here, we studied the biological functions of an FXRα naturally occurring spliced exon 5 isoform (FXRαse5) lacking the second zinc-binding module of the DNA binding domain (DBD). We demonstrate FXRαse5 expression in all FXRα-expressing human and mouse tissues and cells, and that it is unable to bind to its response element or activate FXRα dependent transcription. In parallel, this spliced variant displays differential interaction capacities with its obligate heterodimer partner RXRα that may account for silencing of this permissive dimer for signal transduction. Finally, deletion of exon 5 by gene edition in HepG2 cells leads to FXRα loss-of-function, increased expression of LRH1 metabolic sensor and CD36 fatty acid transporter in conjunction with changes in glucose and triglycerides homeostasis. Together, these findings highlight a novel mechanism by which alternative splicing may regulate FXRα gene function to fine-tune adaptive and/or metabolic responses. This finding deepens our understanding on the role of splicing events in hindering FXRα activity to regulate specific transcriptional programs and their contribution in modifying energy metabolism in normal tissues and metabolic diseases.