Background:Time-restricted feeding (TRF) is a dieting strategy based on nutrients availability and diurnal rhythm, shown to improve lipid metabolism efficiency. We have demonstrated previously that retinoic acid-related (RAR) orphan receptor (ROR)γis the primary transcription factor controlling cholesterol (CHO) biosynthesis program of animals. However, the functional role of RORγin liver physiology of pigs in response to TRF has not been determined, largely due to the lack of functional models and molecular tools. In the present study, we established porcine liver organoids and subjected them to restricted nutrients supply for 10-h during the light portion of the day. Results:Our results showed that TRF regimen did not alter hepatocyte physiology, including unchanged cell viability, caspase 3/7 enzyme activity and the gene signature of cell proliferation in porcine liver organoids, compared to the control group (P>0.05). Furthermore, we found that TRF downregulated the hepatic CHO biosynthesis program at both mRNA and protein levels, along with the reduced cellular CHO content in porcine liver organoids (P<0.05). Using unbiased bioinformatic analysis of a previous ChIP-seq data and ChIP-qPCR validation, we revealed RORγas the predominant transcription factor that responded to TRF, amongst the 12 targeted nuclear receptors (NRs) (P<0.05). This was likely through RORγdirect binding to the MVK gene (encoding mevalonate kinase). Finally, we showed that RORγagonists and overexpression enhanced the enrichment of co-factor p300, histone marks H3K27ac and H3K4me1/2, as well as RNA polymerase Ⅱ (Pol-Ⅱ) at the locus of MVK, in TRF-porcine liver organoids, compared to TRF-vector control (P<0.05). Conclusions:Our findings demonstrate that TRF triggers the RORγ-mediated chromatin remodeling at the locus of CHO biosynthesis genes in porcine liver organoids and further improves lipid metabolism.