Apoptosis is closely interconnected with the severity of renal injury. Magnesium lithospermate B (MLB) extracted from Salviae miltiorrhizae radix showed a wide range of renoprotective effects. However, the underlying mechanisms under beneficial effects have not been elucidated. This study was conducted to investigate whether MLB could mediate renal protection and attenuate apoptosis by mitochondrial pathway. In the present study, the rat model of chronic renal failure (CRF) was established by the 5/6 renal ablation/infarction (A/I) operation. 28 days after the surgery, 30 rats were randomly divided into three groups: sham group, 5/6 (A/I) group and 5/6 (A/I) + MLB group. After 56 days of treatment, we detected the severity of kidney injury, the degree of mitochondrial bax oligomerization and cytochrome c release along with the expression of P53 protein. Our results showed that MLB markedly attenuated kidney injury and apoptosis in 5/6 (A/I) model rats with CRF. MLB effectively inhibited mitochondrial bax accumulation and release of cytochrome c into the cytosol and down-regulated the levels of p53 phosphorylation and acetylation. These data showed that MLB could contribute to renal protection by inhibiting mitochondrial pathway of apoptosis.