The blockade of immune checkpoint molecules such as PD-1 and PD-L1 induces remarkable anti-tumor responses in many cancer patients. These antibodies have been found to significantly prolong survival rate. However, these immunotherapies are not effective in all patients. Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), which can potentially alert host defenses to the presence of danger signals. The alarmin HMGN1 (high-mobility group nucleosome-binding protein) induces DC recruitment and maturation via TLR4 (Toll-like receptor-4). Studies using HMGN1 knockout mice or HMGN1 transfected tumor cells have suggested that HMG proteins play an important role in the generation of anti-tumor immunity in mouse solid tumor models. In this study, we evaluated the anti-tumor effect of systemically administered HMGN1 alone and in combination with anti-PD-L1 monoclonal antibodies. HMGN1 significantly inhibited Colon 26 solid tumor growth at low doses of 0.016–0.4 mg/mouse when administered intraperitoneally in combination with anti-PD-L1 antibody. The anti-tumor effects of HMGN1 were strong and resulted in complete rejection of solid tumors in many cases. Combined HMGN1/anti-PD-L1 antibody treatment also significantly inhibited B16 solid tumor growth. Intraperitoneal administration of mouse HMGN1, human HMGN1 and bovine HMGB1 in combination with anti-PD-L1 Ab showed similar anti-tumor effects. In myeloid differentiation primary response gene 88 (MyD88) knockout mice, no significant changes in tumor volume were observed in HMGN1/anti-PD-L1 antibody treated mice, suggesting that the effects of HMGN1 combination therapy were dependent at least in part on interations between alarmins and TLRs. These findings demonstrate the therapeutic potential of HMGN1 and anti-immune checkpoint antibody combination treatment against cancer.