Earth＇s　rotation　shapes　a　24-h　cycle,　governing　circadian　rhythms　in　organisms.　In　mammals,　the　core　clock　genes,　CLOCK　and　BMAL1,　are　regulated　by　PERIODs　(PERs)　and　CRYPTOCHROMEs　(CRYs),　but　their　roles　remain　unclear　in　the　diamondback　moth,　Plutella　xylostella.　To　explore　this,　we　studied　P. xylostella,　which　possesses　a　simplified　circadian　system　compared　to　mammals.　In　P. xylostella,　we　observed　rhythmic　expressions　of　the　Pxper　and　Pxcry2　genes　in　their　heads,　with　differing　phases.　In　vitro　experiments　revealed　that　PxCRY2　repressed　monarch　butterfly　CLK:BMAL1　transcriptional　activation,　while　PxPER　and　other　CRY-like　proteins　did　not.　However,　PxPER　showed　an　inhibitory　effect　on　PxCLK/PxCYCLE.　Using　CRISPR/Cas9,　we　individually　and　in　combination　knocked　out　Pxper　and　Pxcry2,　then　conducted　gene　function　studies　and　circadian　transcriptome　sequencing.　Loss　of　either　Pxper　or　Pxcry2　eliminated　the　activity　peak　after　lights-off　in　light–dark　cycles,　and　Pxcry2　loss　reduced　overall　activity.　Pxcry2　was　crucial　for　maintaining　endogenous　rhythms　in　constant　darkness.　Under　light–dark　conditions,　1　098　genes　exhibited　rhythmic　expression　in　wild-type　P. xylostella　heads,　with　749　relying　on　Pxper　and　Pxcry2　for　their　rhythms.　Most　core　clock　genes　lost　their　rhythmicity　in　Pxper　and　Pxcry2　mutants,　while　Pxcry2　sustained　rhythmic　expression,　albeit　with　reduced　amplitude　and　altered　phase.　Additionally,　rhythmic　genes　were　linked　to　biological　processes　like　the　spliceosome　and　Toll　signaling　pathway,　with　these　rhythms　depending　on　Pxper　or　Pxcry2　function.　In　summary,　our　study　unveils　differences　in　circadian　rhythm　regulation　by　Pxper　and　Pxcry2　in　P. xylostella.　This　provides　a　valuable　model　for　understanding　circadian　clock　regulation　in　nocturnal　animals.　©　2024　Institute　of　Zoology,　Chinese　Academy　of　Sciences.