A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25mM glucose, but when the concentration of glucose was increased to 35mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.