PD-1 is a type I immune inhibitory transmembrane receptor of the CD28 family that modulates the activity of T cells in peripheral tissues [1]. It is expressed in T cells, B cells, monocytes, natural killer cells and many tumor-infiltrating lymphocytes [2]. Binding of PD-1 to its ligands PD-L1 and PD-L2 reduces T-cell activity [3]. Thereby, under normal conditions, the interaction of PD-1 with PD-L1 or PD-L2 prevents excessive lymphocyte activation and maintains immune tolerance to self-antigens by negatively regulating the immune response [3]. However, PD-L1 is often overexpressed in different tumors including lymphoma, melanoma, nonsmall-cell lung cancer and other types of cancer [2]. As a result, tumor cells attenuate T-cell signaling to evade immune surveillance [4]. Blocking PD-1/PD-L1 interaction has been shown to restore T-cell activation and antitumor response, providing the rationale for therapeutic intervention using PD-1/PD-L1 as target [5]. Currently two monoclonal antibody-based drugs targeting PD-1 are in clinical trials. One is nivolumab or Opdivo from Bristol-Myers Squibb. The other is pembrolizumab or Keytruda, a therapeutic IgG4 antibody developed by Merck. Crystal structures of mouse PD-1 (mPD-1) in complex with human PD-L1 (hPD-L1), mPD-1 complexed with mouse PD-L2 (mPD-L2) and human PD-1 (hPD-1) in complex with hPD-L1 have revealed the structural basis of PD-1’s interaction with its ligands [6-8]. Crystal structure of the full-length pembrolizumab was also reported recently [9]. However, how pembrolizumab specifically recognizes hPD-1 is still unknown. Herein, we report the crystal structure of pembrolizumab Fab (antigen-binding fragment) in complex with hPD-1, revealing the molecular basis for the blockade of hPD-1/hPD-L1 interaction by pembrolizumab.