ObjectiveDifferent serum lipids and lipid-modifying targets should affect the risk of cholelithiasis differently, however, whether such effects are causal is still controversial and we aimed to answer this question.DesignWe prospectively estimated the associations of four serum lipids with cholelithiasis in UK Biobank using the Cox proportional hazard model, including total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG). Furthermore, we estimated the causal associations of the genetically predicted serum lipids with cholelithiasis in Europeans using the Mendelian randomisation (MR) design. Finally, both drug-target MR and colocalisation analyses were performed to estimate the lipid-modifying targets’ effects on cholelithiasis, includingHMGCR,NPC1L1,PCSK9,APOB, LDLR, ACLY, ANGPTL3, MTTP, PPARA, PPARDandPPARG.ResultsWe found that serum levels of LDL-C and HDL-C were inversely associated with cholelithiasis risk and such associations were linear. However, the serum level of TC was non-linearly associated with cholelithiasis risk where lower TC was associated with higher risk of cholelithiasis, and the serum TG should be in an inverted ‘U-shaped’ relationship with it. The MR analyses supported that lower TC and higher TG levels were two independent causal risk factors. The drug-target MR analysis suggested thatHMGCRinhibition should reduce the risk of cholelithiasis, which was corroborated by colocalisation analysis.ConclusionLower serum TC can causally increase the risk of cholelithiasis. The cholelithiasis risk would increase with the elevation of serum TG but would decrease when exceeding 2.57 mmol/L. The use ofHMGCRinhibitors should prevent its risk.