ABSTRACT: Regulatory T cells (Treg) act as a powerful barrier to effective antitumor immunity. Although manipulating Treg is a promising anticancer strategy, doing so while sparing general immune tolerance has been a challenge. Identifying factors specifically expressed in tumor-infiltrating Treg is therefore important for better understanding cancer pathogenesis and identifying novel therapeutic targets that enhance antitumor immunity. We show that T cell Immunoglobulin and Mucin 3 (Tim-3) expression on tumor Treg is required for the function and survival of these cells, in part through Akt and FOXO1 signaling. Deleting Tim-3 in Treg leads to delayed tumor-specific T-cell exhaustion and lower tumor burden, without altering peripheral homeostasis. Similar effects were noted when Tim-3 was only deleted from half of the Treg or when deletion was delayed until after tumor inoculation. Moreover, Treg-specific deletion of Tim-3 cooperated with PD-1 checkpoint blockade to sensitize an immunotherapy-resistant tumor model. In addition, a decrease in Tim-3⁺ tumor Treg correlated with responsiveness to PD-1/LAG-3 combination checkpoint blockade in a human clinical trial. Overall, our data provide evidence that Tim3-expressing Treg are a promising target to modulate tumor-specific immune responses.