[Objective] Generating ribosomal protein SA (RPSA) knockout baby hamster syrian kidney (BHK21) cells using the clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology, and evaluation of the regulatory effect of RPSA on Senecavirus A (SVA) replication. [Methods] We designed 4 pairs of guide RNAs (sgRNA). After screening, the PX330-RPSA-sgRNA2 recombinant plasmid was used for construction of RPSA gene knockout cell line. PX330-RPSA-sgRNA2 was transfected into BHK21 cells, and the monoclonal cell was screened by limited dilution method. The knockout of RPSA was evaluated by Western blotting. The replicative difference of SVA in the wildtype and RPSA knockout cells was investigated by Western blotting and qPCR analysis.[Results] The Western blotting and Sanger sequencing results showed that RPSA within the BHK21 cell line was knocked out. We further investigated the replication of SVA in the RPSA knockout cells which showed that SVA replication was dramatically decreased in RPSA knockout cells comparing with that in the wildtype cells. [Conclusion] The RPSA gene knockout BHK21 cell line was successfully constructed, and RPSA played an important role during SVA replication. The constructed cell line will be a useful tool for exploiting RPSA functions.