Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. OPN is unique as an endogenous negative feedback inhibitor of NO expression. However, the specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)- and NO-mediated induction of OPN synthesis are unknown. We have previously shown that LPS-induced S-nitrosylation of heterogeneous nuclear ribonucleoprotein (hnRNP)-A/B inhibits its activity as a constitutive trans-repressor of the OPN transcription by significantly decreasing its DNA binding activity. hnRNPs were originally described as chromatin-associated RNA-binding proteins that form complexes with RNA polymerase II transcripts. The hnRNP family is comprised of >20 proteins that contribute to the complex around nascent pre-mRNA and are thus able to modulate RNA processing. In this subsequent study, again using RAW 264.7 murine macrophages and COS-1 cells, we demonstrate that hnRNP-A/B and hnRNP-U proteins serve antagonistic transcriptional regulatory functions for OPN expression in the setting of LPS-stimulated NO synthesis. In the presence of NO, hnRNP-A/B dissociates from its OPN promoter site with subsequent derepression of OPN promoter activity. Subsequently, hnRNP-U binds to the same site to further augment OPN promoter activation. This has not been previously described for the hnRNP proteins. Our results represent a unique transcriptional regulatory mechanism which involves interplay between members of the hnRNP protein family.