The present study was aimed to determine whether stimulating Npr1 gene activity using Valporic acid (VA), a small short chain fatty acid molecule can enhance ANP mediated anti-hypertrophic activity in isoproterenol (ISO) – treated H9c2 cells in vitro. H9c2 cells were treated with ISO (10−5 M) and co-treated with VA (10−5 M) in the presence and absence of ANP (10−8 M), for 48 h. ATRA (10−5 M) was used as a positive inducer of Npr1 gene transcription. The mRNA expression of Npr1 and PKG-I genes, proto-oncogenes (c-fos, c-jun and c-myc) and hypertrophic markers (ANP, BNP, α-sk and β-MyHC), genes were determined by quantitative PCR (qPCR). The protein profiling of NPR-A, PKG-I and cGMP were evaluated by Western blot, immunofluorescence and ELISA respectively. A marked reduction in the level of expression of Npr1 (3- fold) and PKG-I (2.5-fold) genes and increased expression of proto-oncogenes (p< 0.001, respectively) and hypertrophic marker genes (p<0.001, respectively) were noticed in the ISO-treated H9c2 cells as compared with control cells. In contrast, the VA treated cells showed maximal Npr1 gene expression (3.5-fold) as compared with ATRA treated cells (2 fold), which is well correlated with the intracellular cGMP levels (80% vs 60%) and reduced (2.5-fold) HDAC −1&−2 mRNA expression. Furthermore, VA or ATRA treatment effectively reversed the ISO-induced altered expression of Npr1 and PKG-I genes, proto-oncogenes, and hypertrophic markers genes. Interestingly, the results of the present study suggest that ANP mediated anti-hypertrophic activity was enhanced with either VA (p<0.001) or ATRA (p<0.01) co-treatment. Together, we conclude that VA in combination with ANP can be a novel therapeutical approach for the treatment and management of left ventricular cardiac hypertrophy.