Aim:　The　persistently　high　mortality　rate　of　pancreatic　ductal　adenocarcinoma　(PDAC)　is　largely　attributed　to　the　acquired　resistance　to　chemotherapy,　particularly　gemcitabine.　This　study　aims　to　elucidate　the　underlying　molecular　mechanisms　of　gemcitabine　resistance　in　PDAC,　uncover　additional　pro-tumorigenic　factors　contributing　to　drug　resistance,　and　develop　more　effective　and　safer　targeted　therapeutic　strategies　against　this　phenomenon.　Methods:　Circular　RNA　(circRNA)　sequencing　was　employed　to　identify　differentially　expressed　circRNAs　between　chemo-sensitive　and　resistant　tumors.　Liquid　Chromatography-Mass　Spectrometry　(LC-MS)　was　utilized　to　uncover　the　RNA-binding　proteins　(RBPs)　associated　with　circular　RNA　of　alpha-1,　3-glucosyltransferase　8　(cALG8).　Molecular　biology　techniques　were　applied　to　explore　the　biological　functions　and　regulatory　mechanisms　of　cALG8　in　the　context　of　gemcitabine　resistance　in　PDAC.　Single-cell　sequencing　was　performed　to　reveal　changes　in　the　composition　of　tumor　immune　microenvironment　of　pancreatic　cancer.　Patient-Derived　Organoid　(PDO)　and　Patient-Derived　Xenograft　(PDX)　were　employed　to　further　validate　the　molecular　mechanisms.　Finally,　antisense　oligonucleotides　(ASOs)　targeting　cALG8　were　developed　for　in　vivo　use,　and　their　translational　therapeutic　potential　was　evaluated　in　mouse　models.　Results:　This　study　identified　that　cALG8,　which　is　associated　with　alternative　splicing,　is　highly　expressed　in　gemcitabine-resistant　PDAC　cells.　cALG8　regulates　the　alternative　splicing　complex,　thereby　promoting　chemoresistance　and　immunosuppression　in　PDAC.　Mechanistically,　high　level　of　cALG8　functions　as　a　protein　scaffold　through　its　34-85　nt　and　109-160　nt　regions,　creating　spatial　conditions　for　CDC-like　kinase　1　(CLK1)　to　phosphorylate　serine/arginine-rich　splicing　factor　7　(SRSF7)　at　site　231S.　This　process　facilitates　the　formation　of　the　SRSF7-dependent　ataxia-telangiectasia　mutated　(ATM)　kinase　variant,　ATM203,　enhancing　the　translational　efficiency　of　ATM,　and　consequently　promoting　DNA　damage　repair　and　immune　microenvironment　remodeling　in　PDAC　cells　to　counteract　the　effects　of　chemotherapeutic　drugs.　A　cALG8-targeting　ASO　that　disrupts　the　CLK1-SRSF7　interaction,　when　combined　with　gemcitabine　and　anti-programmed　cell　death　protein　(PD)-1　antibody,　significantly　reduced　tumor　burden　in　PDX　model,　validating　its　therapeutic　translational　value.　Conclusion:　We　demonstrated　that　the　cALG8/CLK1/SRSF7　axis　promotes　ATM　expression　by　enhancing　the　splicing　of　ATM203,　thereby　facilitating　gemcitabine　resistance　and　formation　of　an　immunosuppressive　microenvironment　in　PDAC.　This　insight　aids　in　the　development　of　drugs　targeting　chemotherapy　resistance　induced　by　DNA　damage　repair　mechanisms　in　PDAC.