Hepatocellular carcinoma (HCC) is one of the most aggressive types of cancer and currently lacks effective treatment strategies. The present study revealed that deoxyribonuclease 1 like 3 (DNase1L3) expression levels were significantly downregulated in numerous types of gastrointestinal cancer, and especially in HCC. Tissue microarrays were further used to illustrate that DNase1L3 expression levels were frequently downregulated in HCC tissues compared with normal liver tissues. In addition, DNase1L3 expression levels were identified to be significantly associated with tumor size (p=0.0028), tumor thrombus formation (p<0.01), and a poorer overall survival (p=0.005) and disease-free survival (p=0.006) of HCC. Gene Ontology functional term enrichment analysis of biological processes discovered that DNase1L3 was significantly associated with complement activation. Further studies demonstrated that the ectopic expression of DNase1L3 suppressed cell growth and inhibited the PI3K/AKT signaling pathway activation following C3a receptor agonist treatment. In conclusion, the findings of the present study suggested, for the first time, that DNase1L3 may serve as a biomarker for the prognosis of patients with HCC, and may suppress HCC growth via inhibiting the PI3K/AKT signaling pathway.