Liver receptor homologue-1 (LRH1) is an orphan nuclear receptor that has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the LRH1 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative LRH1 co-regulators. Nuclear fractionation protocol was essential for detection of LRH1 peptides by mass spectrometry (MS), with most peptides being observed in the insoluble fraction (receptor bound to DNA). SERBP1 and ILF3 were identified as LRH1 interacting partners by both Western blot and MS/MS analysis. Receptor knockdown by siRNA showed an increase in SERBP1 expression, while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of LRH1 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, subsequently inhibiting transcription. Given the receptor's role in cancer progression, the study here elucidates additional transcriptional machinery involved in LRH1 signaling and potentially provides new targets for therapeutics development.