Aim. To investigate the genetic contribution of adenosine A3 receptor (ADORA3) gene polymorphisms in the pathogenesis of chronic heart failure (CHF). Methods. Firstly, a case-control study was performed to investigate the association of ADORA3 polymorphisms with CHF risk. Three hundred northern Chinese Han CHF patients and 400 ethnicity-matched healthy controls were included. Four polymorphisms were genotyped. This case-control study was also replicated in 304 CHF patients and 402 controls from southern China. Finally, the functional variability of positive polymorphism was analyzed using luciferase reporter assay and real-time PCR. Results. Overall, the rs1544223 was significantly associated with CHF risk under the dominant model (P=0.046, OR = 1.662, 95% CI = 1.009–2.738). But it did not affect disease severity. These results were also consistent in replicated population. In addition, the transcriptional activity for promoter with the A allele was lower than that with the G allele (n=3, 4.501±0.308 versus 0.571±0.114, P<0.01) and ADORA3 mRNA levels were significantly higher in GG homozygotes than subjects carrying GA (n=6, 0.058±0.01 versus 0.143±0.068, P=0.004) or AA genotypes (n=6, 0.065±0.01 versus 0.143±0.068, P=0.008). Conclusions. Should the findings be validated by further studies with larger patient samples and in different ethnicities, they may provide novel insight into the pathogenesis of CHF.