Thymosin alpha 1 (T alpha 1) has been tested for cancer therapy for several years, in most cases, the anti-tumor effect of T alpha 1 was limited, especially when T alpha 1 was used as a single agent. The role of T alpha 1 in cancer treatment and the regulatory mechanisms by which T alpha 1 takes effects are not yet completely understood. Using a Lewis lung caner model, here we report that T alpha 1 used alone elevated CD8(+) T cells, but failed to inhibit tumor growth. Furthermore, immunosuppressive myeloid-derived suppressor cells (MDSCs) showed heightened Arginase 1 production in response to T alpha 1 treatment, which led to stronger suppression of anti-tumor immunity. In addition, the upregulation of ARG1 was dependent on TLR5/MyD88 signaling, blocking MyD88 signaling abrogated the enhanced ARG1 expression and restored the antitumor efficacy of T alpha 1. This study provides the first demonstration that T alpha 1 treatment activates but not expands MDSCs via MyD88 signaling, which indicates better immunotherapeutic strategy of T alpha 1 against cancer. (C) 2015 Elsevier Inc. All rights reserved.