The expression of human Parvovirus B19 nonstructural protein 1 (NS1) induces cell cycle arrest at the G1 phase and is accompanied by increased expression of the cyclin-dependent kinase inhibitor, p21/WAF1. Here, we provide direct evidence that NS1 mediates the transactivation of p21/WAF1. Up-regulation of p21/WAF1 by wild-type NS1 but not an NS1 mutant deleted of its NTP binding motif was observed. We also demonstrated that the wild-type NS1 is unable to induce G1 arrest in p21-deficient cells. Using reporter plasmids containing various mutants of the p21/WAF1 promoter, luciferase assay further revealed that the binding sites of the promoter to the transcription factor Sp1 are critical for NS1-mediated transactivation. Indeed Sp1 interacts only with the wild-type NS1 but not the NS1 mutant. These results indicate a cooperative contribution of NS1 and Sp1 to the transactivation of p21/WAF1, which leads to G1 arrest.