Silique number is the most important component of yield in rapeseed ( Brassica napus L.). To dissect the mechanism underlying the natural variation of silique number in rapeseed germplasm, a series of studies were performed. A panel of 331 core lines was employed to genome‐wide association study ( GWAS), and 27 loci (including 20 novel loci) were identified. The silique number difference between the more‐ and fewer‐silique lines can be attributed to the accumulative differences in flower number and silique setting rate. Each of them accounted for 75.2% and 24.8%, respectively. The silique number was highly associated with the total photosynthesis and biomass. Microscopic analysis showed that the difference between extremely more‐ and fewer‐silique lines normally occurred at the amount of flower bud but not morphology. Transcriptome analysis of shoot apical meristem ( SAM) suggested that most of enriched groups were associated with the auxin biosynthesis/metabolism, vegetative growth and nutrition/energy accumulation. By integrating GWAS and RNA‐seq results, six promising candidate genes were identified, and some of them were related to biomass accumulation. In conclusion, the natural variation of silique number is largely affected by the biomass and nutrition accumulation, which essentially reflects the positive regulatory relationship between the source and sink. Our study provides a comprehensive and systematic explanation for natural variation of silique number in rapeseed, which provides a foundation for its improvement.