The RNA-binding protein PNO1 is critical for ribosome biogenesis, but its potential role in cancer remains unknown. In the present study, on-line data mining, cDNA and tissue microarrays indicated that PNO1 expression was higher in colorectal cancer (CRC) tissue than in noncancerous tissue, and its overexpression was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated that PNO1 knockdown suppressed growth of CRC cells in vitro and in vivo, while PNO1 overexpression promoted CRC cell proliferation in vitro. In CRC cells expressing wild-type p53, PNO1 knockdown enhanced expression of p53 and its downstream gene p21, and reduced cell viability; these effects were prevented by p53 knockout and attenuated by the p53 inhibitor PFT-α. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, of 40S and 60S ribosomal subunits, and of the 80S ribosome. It also reduced global protein synthesis, increasing nuclear stress and inhibiting MDM2-mediated ubiquitination and p53 degradation. Overexpressing EBF1 suppressed PNO1 promoter activity and decreased PNO1 mRNA and protein, inhibiting cell proliferation and inducing cell apoptosis through the p53/p21 pathway. In CRC tissues, the expression of EBF1 correlated inversely with PNO1. Data mining of online breast and lung cancer databases showed increased PNO1 expression and association with poor patient survival; PNO1 knockdown reduced cell viability of cultured breast and lung cancer cells. Taken together, these findings indicate that PNO1 is overexpressed in CRC and correlates with poor patient survival, and that PNO1 exerts oncogenic effects, at least in part, by altering ribosome biogenesis.