The oil yield of Brassica napus is determined by both seed yield and oil content. A previous study showed that strong RNA interference, designed to simultaneously knock down all four BnAP2 paralogs, reduced seed yield due to abnormal floral development in B. napus. However, the function of individual, specific BnAP2 paralogs in seed oil production and the underlying mechanisms remain unclear. Here, we found that all four BnAP2 paralogs present in the genome of the B. napus ‘K407’ inbred line were highly expressed in flowers and developing seeds; however, BnaA01.AP2 was minimally expressed in floral tissues. Interestingly, BnaA01.AP2 knockout mediated by CRISPR/Cas9 resulted in significant increases in both seed yield and oil content, thereby increasing overall seed oil yield without detectable negative effects on the other examined agronomic traits. Furthermore, we demonstrated that BnaA01.AP2 repressed oil accumulation by directly downregulating BnaA09.WRI1, BnaA03.BCCP1, BnaA09.L1L, and BnaC09.L1L and indirectly regulating a series of key genes involved in glycolysis, fatty acid biosynthesis, and triacylglycerol assembly during B. napus seed development. Our research not only provides insights into the regulatory mechanisms of seed oil accumulation but also provides promising genetic resources and germplasm for breeding cultivars with higher seed oil yield in B. napus.