Previous reports have suggested that many gut microbiomes were associated with the development of colorectal cancer (CRC), and could modulate response to numerous forms of cancer therapy, including checkpoint blockade immunotherapy. Here we evaluated the protective efficacy of Lactobacillus acidophilus (L. acidophilus) cell lysates combined with an anti-CTL antigen-4 blocking antibody (CTLA-4 mAb) in syngeneic BALB/c mice CRC models induce by a single intraperitoneal injection of 10 mg/kg azoxymethane (AOM), followed by three cycles of 2% dextran sulfate sodium (DSS) in drinking water. In contrast to CTLA-4 mAb monotherapy, L. acidophilus lysates could attenuate the loss of body weight and the combined administration significantly protected mice against CRC development, which suggested that the lysates enhanced antitumor activity of CTLA-4 mAb in model mice. The enhanced efficacy was associated with the increased CD8 + T cell, increased effector memory T cells (CD44 + CD8 + CD62L+), decreased Treg (CD4 + CD25 + Foxp3+) and M2 macrophages (F4/80 + CD206+) in the tumor microenvironment. In addition, our results revealed that L. acidophilus lysates had an immunomodulatory effect through inhibition the M2 polarization and the IL-10 expressed levels of LPS-activated Raw264.7 macrophages. Finally, the 16S rRNA gene sequencing of fecal microbiota demonstrated that the combined administration significantly inhibited the abnormal increase in the relative abundance of proteobacteria and partly counterbalance CRC-induced dysbiosis in model mice. Overall, these data support promising clinical possibilities of L. acidophilus lysates with CTLA-4 mAb in cancer patients and the hypothesis that probiotics help shape the anticancer immune response.