The second open reading frame of avian reovirus S1 gene segment encodes a 17 kDa non-structural protein, named p17. The biological role of p17 is fully unknown so far. Using trypan blue dye exclusion and MTT assay, we demonstrated that the ectopic expression of p17 results in the reduction of viable cell number and cell proliferation rate of Vero, BHK, 293, and HeLa cells. Measurement of LDH activity and DNA fragmentation analysis revealed that p17 expression did not cause cell death or apoptosis. These data indicated that the p17 possessed the growth retardation function. Semi-quantitative RT-PCR and Western blotting revealed that p17-expressing cells induced the expression of CDK inhibitor p21cip1/waf1 in a time- and dose-dependent manner, but the transcripts of CDK inhibitor p15INK4b, p16INK4a, or p27kip were not altered. In the presence of p17, the p53 protein level and p53-driven reporter activity were elevated significantly. Dominant negative p53 alleviated the p21 accumulation, p53 activation, and growth inhibition effect induced by p17. Taken together, these studies revealed a possible intrinsic function of p17 in growth regulation through the activation of p53 and p21cip1/waf1.