Ribosome biogenesis is a fundamental cellular process and occurs mainly in the nucleolus in eukaryotes. The process is exceptionally complex and highly regulated by numerous ribosomal and non-ribosomal factors. A recent discovery strengthened the link between ribosome biogenesis and malignant transformation. Here, we determined that Nop-7-associated 2 (NSA2) is a nucleolar protein required for ribosome biogenesis. NSA2 knockdown reduced the rate of rRNA synthesis, diminishing the 60S ribosomal subunit. Moreover, we demonstrated that depletion of NSA2 suppressed protein synthesis. To investigate the signaling pathway affected by NSA2, NSA2 was depleted, which triggered the inactivation of the mTOR signaling pathway. Taken together, our findings reveal a novel function of NSA2 and provide insight into the regulation of ribosome biogenesis by NSA2.