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<h3>Background</h3> Colorectal cancer (CRC) is the third most common cancer diagnosed in both men and women, with the majority of patients dying from hepatic metastasis. Surgery and chemotherapy are standard treatments for colorectal liver metastasis (CRCLM). However, resection is feasible in only 10–20% of cases, with a 5-year survival rate as low as 30%. Immune checkpoint inhibitors (ICIs), which target inhibitory receptors on T cells, have become a therapeutic option for CRCLM, especially in deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) tumors. However, ICIs have not shown significant clinical success in the majority (~85%) of CRCs, which are MMR proficient (pMMR)/microsatellite stable (MSS). MMR integrity is often assessed by evaluating the expression of four MMR proteins: MLH1, PMS2, MSH2, and MSH6. Complete absence of any of these proteins indicates dMMR. Our recent findings show that neutrophil extracellular traps (NETs) form in the livers of mice with CRCLM, and destroying NETs with DNase I significantly reduces the growth of metastatic liver tumors in syngeneic CRC models of MSI-H/dMMR (MC38). However, the role of NETs in pMMR/MSS CRCLM progression is unknown. <h3>Methods</h3> For pMMR/MSS CRCLM tumor models, one million CT26 murine CRC cells were injected into the portal vein of mice using a 30-G needle. Tumor growth was measured by in vivo bioluminescent imaging weekly, and gross tumor nodules were counted upon mouse sacrifice three weeks after surgery. Anti-PD-1 or IgG2a isotype antibody (10 mg/kg, every 3 days) and DNase I (5 mg/kg, daily) were administered intraperitoneally starting from day 5 after tumor cell inoculation. Tumor growth was determined weekly by bioluminescence imaging. Bulk RNA sequencing (RNA-seq) for tumor cells, excluding CD45+ immune cells, was conducted. CD45+ immune cell populations and CD8+ T cell function in the tumor were quantified by flow cytometry. <h3>Results</h3> DNase I decreases tumor-associated neutrophils and NET formation in vivo, thereby significantly reducing resistance to anti-PD-1 blockade in our mouse pMMR/MSS CRCLM model by attenuating tumor growth. Mechanistically, inhibiting NETs with DNase I not only reverses anti-PD-1 blockade resistance by reducing MSH2 and MSH6 expression in tumor cells but also increases CD8+ T cell infiltration and cytotoxicity compared to anti-PD-1 treatment alone. <h3>Conclusions</h3> These findings represent a novel approach to targeting the tumor microenvironment using DNase I, alone or in combination with immune checkpoint inhibitors.