Despite advances in therapy, cancer continues to be a major health challenge in the 21st century, with mortality rates still hovering around approximately 20%. The tumor suppressor p53, a protein critical for cancer development and prevention, is regulated by various interacting partners. In our previous unpublished findings, we identified that NR5A1 (also called steroidogenic factor-1, SF-1, the master regulator for adrenal and gonadal steroidogenesis) is able to module p53 activity. Consequently, in this investigation, we broaden our exploration to examine how NR5A1 regulates p53 transactivation using the p53(14X)RE-LUC system. Generally, H1299 (p53 NULL) cells in a 24-well plate were transfected with the p53(14X)RE-LUC plasmid and with or without different expression plasmids by the Fugene HD transfection method. After 48 hours of transfection, luciferase activities were analyzed by the Dual-Luciferase Reporter System. Western blot analysis was analyzed for protein expression. We first found that NR5A1 enhances wild-type (WT) p53 transactivation in a dose-dependent manner in H1299 cells. This result is confirmed by testing JDP2 (we reported earlier that JDP2 activates WT p53 transactivation) and NR5A1 in the same experiments. Given that MDM2 is the major negative regulator of p53, we then tested whether NR5A1 could counteract MDM2-mediated p53 repression. We found that NR5A1 reverses MDM2-mediated p53 transactivation, suggesting that NR5A1 may have the ability to bind to both p53 and MDM2 in order to abolish MDM2-mediated p53 repression. Finally, we found that NR5A1 increases wild-type (WT) p53, but not G245S (hotspot mutation) p53, transactivation. Taken together, our preliminary results demonstrate that NR5A1 is a novel protein that regulates p53 activity likely involving MDM2 competition due to the possible p53-NR5A1 interaction. This data substantiates the concept that p53’s activity is governed by a complex and still-expanding network of interactions.