Objective To explore whether the combination of bortezomib and Bcl-2 inhibitor (obatoclax, AT-101, ABT-199) can synergistically induce the apoptosis of human acute B lymphoblastic leukemia cell line Nalm-6. Methods MTT assay was used to evaluate cell viability of Nalm-6 cells in response to Bcl-2 inhibitor alone or combined treatment for 48 h. Apoptosis was examined by flow cytometry and the expression of Bcl-2 family proteins, ubiquitin, microtubule-associated protein 1 light chain 3B (LC3B), p62, binding immunoglobulin protein (Bip), phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and C/EBP homologous protein (CHOP) was detected by Western blotting after drug alone or combined treatment. The mRNA levels of critical factors of endoplasmic reticulum stress (ERS)response, including Bip, CHOP, activating transcription factor (ATF) 4, ATF6, inositol-requiring enzyme 1α (IRE1α) and X-box binding protein 1 (XBP1) were measured by qRT-PCR. Finally, MTT and flow cytometry were used to determine whether tauroursodeoxycholate acid (TUDCA, an ERS inhibitor) could reverse the apoptosis induced by the combination of the two drugs. Results The application of bortezomib, obatoclax, AT-101 and ABT-199 alone reduced the viability of Nalm-6 cells. Obatoclax potentiated the cytotoxicity of Nalm-6 cells in response to bortezomib, but not including AT-101 or ABT-199. Obatoclax blocked autophagy flux by upregulating the protein expression of LC3B-Ⅱ and p62. The accumulation of ubiquitin protein was observed after use of bortezomib or obatoclax alone, but the protein significantly increased after two drug combination. Bortezomib combined with obatoclax caused the dual blockade of autophagy and proteasome and a large amount of protein accumulation, leading to activated ERS, finally to cell apoptosis. TUDCA reduced the apoptosis induced by two drug combination. Conclusion Bortezomib in combination with obatoclax can simultaneously inhibit autophagy and protease activity, triggering ERS, finally inducing human acute B lymphoblastic leukemia cell apoptosis.