Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most severe diseases affecting the growth and production of Brassica napus. Sphingolipid metabolism plays a crucial role in plant response to pathogens. In this study, we show that ceramide kinase (CERK) is significantly induced during S. sclerotiorum infection to produce higher levels of ceramide-1-phosphate (C1P) in B. napus. The balance between ceramide (Cer) and C1P affects plant resistance to S. sclerotiorum, with CERK mutant lines exhibiting greater susceptibility to S. sclerotiorum and CERK overexpression lines showing enhanced resistance to this pathogen. Moreover, we identified candidate C1P-binding proteins by proteomic analysis and determined that C1P binds to and promotes the activity of a Gly-Asp-Ser-Leu lipase protein involved in B. napus resistance to S. sclerotiorum infection. In conclusion, our results indicate that C1P plays a key role in S. sclerotiorum resistance through metabolic regulation and signal transduction in B. napus.