Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intratumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor, and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab')2, and a full antibody, anti-CD25-IgG, as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab')2-IR700 i.v. with NIR light exposure; and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab')2-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab')2-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on the activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab')2 based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.