Objective To explore the role of protein kinase 9 (PK9) in the growth and development of Plasmodium yoelii. Methods The PK9 sequence information of P. yoelii lethal strain 17XL (Py17XL) was obtained from PlasmoDB. The sequence homology of PK9 among P. yoelii (PyPK9), P. falciparum (PfPK9) and human protein kinase p78 was compared by BLAST. The primers were designed based on the PyPK9 DNA sequence and coding DNA was amplified from WT Py17XL genomic DNA. The homologous arm and sgRNA sequence were ligated into PYC-MCS plasmid by CRISPR-Cas9 technique to construct PK9 gene knockout (PK9KO) recombinant plasmid. The recombinant plasmid was transferred into Py17XL by electroporation, screened and cloned by pyrimidine. The recombinant plasmid was identified by PCR and DNA sequencing. To determine the virulence of erythrocytic stage of PK9KO P. yoelii, 5 female BALB/c mice were intraperitoneally infected with 1 × 10⁵ PK9KO P. yoelii erythrocytic stage. Another 5 mice were infected WT P. yoelii as control. Blood was taken every 24 h to detect parasitemia in mice, and the survival of the mice was observed(3 repeated experiments). To determine the development of PK9KO P. yoelii in mosquito, 200 Anopheles stephensi mosquitoes aged 3 to 5 days were randomly divided into two groups: WT group (n = 100) and PK9KO group (n = 100), each was infected with WT P. yoelii or PK9KO P. yoelii through feeding with infected mice. The mosquito stomach was dissected 8 days after being fed with blood, Each group was randomly selected from 15 Anopheles stephensi for necropsy, and the number of the developed oocysts in mosquito stomach was examined. To determine the infectivity of PK9KO P. yoelii sporozoites developed in infected mosquitoes, 10 female BALB/c mice were randomly divided into WT sporozoite infection group (n = 5) and PK9KO sporozoite infection group (n = 5). Each mouse was infected with 1 × 10³ sporozoites through intravenous injection(2 repeated experiments). Tail vein blood was taken every 12 hours after injection, and the occurrence time of parasitemia was observed by blood smear. Results The sequence homology of PyPK9 with PfPK9 was 82.5%, but only 39.4% with human protein kinase p78. The PCR product of PK9 of P. yoelii was 597 bp. The PK9KO recombinant plasmid was constructed and the PK9 gene was successfully knocked out in Py17XL strain conformed by PCR identification and DNA sequencing. All the mice died on the 5th to 6th day after being infected with WT P. yoelii, however, all mice infected with PK9KO P. yoelii were survived and the parasitemia disappeared on the 17 d after infection. However, there was no significant difference for the infection of mosquito (formation of oocyst) by feeding with mouse blood with WT P. yoelii (60.0%, 9/15) and with PK9KO P. yoelii (66.7%, 10/15) (P > 0.05). The infectivity of sporozoites developed from WT and PK9KO P. yoelii remained similar in infected mice. Parasitemia was found 72 h after intravenous infection of both sporozoites. Conclusion PK9 plays an important role in the virulence of erythrocytic stage of P. yoelii.