Background and Purpose: Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the world’s population. The development of NAFLD affects the morphological structure, number and physiological function of liver mitochondria. Corydalis Saxicola Bunting Total Alkaloids (CSBTA), a promising therapeutic candidate for NAFLD, can play the role of anti-inflammation and hepatoprotection. The present study aimed to investigate the protective effect of CSBTA and its underlying mechanism. Experimental Approach: Our group used high fat and high cholesterol diet (HFHCD) to feed C57BL/6 mice for 20 weeks to establish a NAFLD disease model. CSBTA was administered at week 12 for 8 weeks. Palmitic acid (PA) was co-cultured with AML12 cells for 24 hours to establish lipid accumulation model in vitro. Histopathology, biochemical indicators of lipid metabolism, function of hepatocellular mitochondrial were detected and gene expression of mitochondrial biogenesis and mitophagy were also evaluated. Key Results: The results indicated that CSBTA can alleviate HFHCD and PA induced lipid accumulation, improve hepatocellular mitochondrial structure and function. CSBTA upregulated the gene and protein expression related to mitochondrial biogenesis, increased the content of mtDNA and promoted the normal proceed of mitophagy in damage cells, which blunted an effective operation of CSBTA on ameliorating NAFLD. Conclusion and Implications: CSBTA can effectively maintain the mitochondrial function in vivo and in vitro, increase the expression levels of mitochondrial biogenesis related genes in liver and AML12 cells, promote the occurrence of mitophagy, maintain the quantitative and qualitative homeostasis of mitochondria in the body, and ultimately improve NAFLD.