Aristolochic acid I (AAI) is one of the nephrotoxic and carcinogenic compounds in Aristolochic acids (AAs). Recent studies have reported its promoting effect on hepatocellular carcinoma. However, the underlying mechanisms of AAI for the development of HCC is still unclear. Here, we found that AAI exposure caused alterations in mitochondrial function, which featured with increased ATP level and mitochondrial membrane potential, accumulation of mitochondrial Ca2+ and mitochondrial ROS (MitoROS) in Hepa1-6 and HepG2 cells. The restriction of mitochondrial Ca2+ uptake alleviated these effects. Our results showed that increased MitoROS was associated with AAI-induced migration and invasion in HCC cells. MitoROS/Src/FAK pathway was involved in the AAI-induced migration and invasion of HCC cells. In summary, our study showed that AAI affected mitochondrial metabolism of HCC cells by promoting the accumulation of mitochondrial Ca2+. These effects resulted in the activation of the MitoROS/SRC/FAK pathway in AAI-treated HCC cells, which in turn induced cell migration and invasion.