Aim To investigate the effects of hyperglycemia on normoxia and hypoxia apoptosis in primary neonatal rat cardiac myocytes. Methods To culture the primary neonatal rat cardiac myocytes and exposure to normoxia and hypoxia environment in medium containing 33 mmol/L glucose and 5.5 mmol/L glucose (control). The hypoxia environment was achieved by treating cells with cobalt chloride. Cell viability was measured by Trypan blue staining. Cardiomyocyte apoptosis was detected with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and Annexin V/PI binding assay. Results In 33 mmol/L glucose group cell survival rate was lower (P<0.01) and apoptosic rate was higher (P<0.01) than those in 5.5 mmol/L glucose group in normoxia condition. However, in hypoxia condition, cell survival rate was higher (P<0.01) and apoptosic rate was lower (P<0.05) in 33 mmol/L glucose group than those in 5.5 mmol/L glucose group. Conclusion In cultured primary neonatal rat cardiac myocytes, hyperglycemia induced apoptosis in normxia condition. However, hyperglycemia plays an anti-apoptotic protective role in cobalt chloride mimic chemical hypoxia condition.