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The kinesin-9 family members Kif6 and Kif9 are implicated in motile cilia motilities across protists and mammals. How they function and whether they act redundantly, however, remain unclear. Here, we show that Kif6 and Kif9 play distinct roles in mammals. Kif6 forms puncta that move bidirectionally along axonemes, whereas Kif9 appears to oscillate regionally on the ciliary central apparatus. Consistently, only Kif6 displays microtubule-based motor activity in vitro, and its ciliary localization requires its ATPase activity. Kif6 deficiency in mice disrupts coordinated ciliary beat across ependymal tissues and impairs cerebrospinal fluid flow, resulting in severe hydrocephalus and high mortality. Kif9 deficiency causes mild hydrocephalus without obviously affecting the ciliary beat or the lifespan. Kif6\u2212\/\u2212 and Kif9\u2212\/\u2212 males are infertile but exhibit oligozoospermia with poor sperm motility and defective forward motion of sperms, respectively. These results suggest Kif6 as a motor for cargo transport and Kif9 as a central apparatus regulator.<\/jats:p>","DOI":"10.1083\/jcb.202312060","type":"journal-article","created":{"date-parts":[[2024,7,31]],"date-time":"2024-07-31T02:21:11Z","timestamp":1722392471000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":12,"title":["Distinct roles of Kif6 and Kif9 in mammalian ciliary trafficking and motility"],"prefix":"10.1083","volume":"223","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-0903-8634","authenticated-orcid":true,"given":"Chuyu","family":"Fang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rrdvm96","id-type":"ROR","asserted-by":"publisher"}],"name":"Chinese Academy of Sciences 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, , Shanghai, China"},{"name":"University of Chinese Academy of Sciences 2 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