Ribosomal protein L21 (RPL21) plays an important role in ribosome assembly. It is considered to be a major cause for the occurrence of the hypotrichosis simplex (HTS), a type of sustained hair loss from early childhood to adulthood. In this study, the full-length sequence of pig RPL21 gene (GenBank accession number: KU891824) was cloned and identified for the first time. We found it contains a 483-bp open reading frame (ORF) encoding 160 amino acids. It is located in the plus strand of chromosome 11, which spans 2,167 bp from 4,199,792 to 4,201,958. We found RPL21 expression level is closely related to cell proliferation and cell cycle arrest. In the knockdown group, the cell proliferation activity was significantly decreased (P<0.01) and an obvious accumulation of cells at the G2/M phase with a simultaneous up-regulation of p53 and p21 was observed. This likely due to knockdown of RPL21 triggered ribosomal stress, which affected the normal ribosome assembly and caused defective ribosome biogenesis. The unassembled RPs were released consequently from the nucleolus to the nucleoplasm where they can activate p53-dependent cell-cycle responsive factors and led to a G2/M arrest. We expect these results may provide valid information for further study on the pig RPL21 gene and the cause of hypo trichosis simplex.