Programmed death-1 (PD-1, CD279), an inhibitory co-stimulatory molecule of the CD28 superfamily, plays a critical role in immune response. In this report, four novel mouse anti-human PD-1 monoclonal antibodies (MAbs) were prepared using hybridoma technology and immunological characteristics of the MAbs were determined. The results showed that all the MAbs (clones 9H1, 4B9, 8F5, and 1F8) were IgG1(kappa) and bound specifically to human PD-1. By mutual competition, we found that the antibodies recognized three different epitopes of PD-1 antigen and 9H1 MAb could block both PD-1/PD-L1 and PD-1/PD-L2 interaction. Cross-linking of PD-1 with MAb 9H1 markedly blocked PD-1 negative signal and promoted T cell proliferation. In addition, 4B9 and 9H1 MAbs were suitable for indirect ELISA detection. Thus, the MAbs against human PD-1 with high specificity and different activity would be useful for the further study of this molecule.