AbstractLiver cancer is a prevalent malignant tumor with high mortality worldwide, making it urgent to explore new targets for liver cancer therapy. N-terminal EF-hand calcium binding protein 3 (NECAB3) is a new recognized regulator of cancer, while its role in liver cancer remained elusive. Thus, the study clarified the action of NECAB3 on liver cancer development and explored the detailed mechanism. We found that NECAB3 was enhanced in liver cancer. Knockdown of NECAB3 restrained liver cancer cell migration and invasion. Besides, knockdown of NECAB3 suppressed the activation of the hypoxia-inducible factor 1-alpha (HIF-1α)/Ras like without CAAX 1 (RIT1) pathway. Furthermore, NECAB3 regulated liver cancer migration and invasion through modulating RIT1 expression. Moreover, downregulation of NECAB3 suppressed liver cancer tumor growthin vivo. In conclusion, NECAB3 was upregulated in liver cancer. Knockdown of NECAB3 suppressed aggressive phenotype of liver cancer via modulating the HIF-1α/RIT1 axis, providing a possible target for liver cancer therapy.