Rapeseed (Brassica napus), an important oil crop worldwide, provides large amounts of lipids for human requirements. Calcineurin B-like (CBL)-interacting protein kinase 9 (CIPK9) was reported to regulate seed oil content in plant. Here, we generated gene-silenced lines through RNA interference biotechnology and loss-of-function mutant bnacipk9 using CRISPR/Cas9 to further study BnaCIPK9 functions in seed oil metabolism of rapeseeds. We discovered that compared with wild type (WT) lines, gene-silenced and bnacipk9 lines had substantially different oil contents and fatty acid compositions: seed oil content was improved by 3%–5% and 1%–6% in bnacipk9 lines and gene-silenced lines, respectively; both lines were with increased levels of monounsaturated fatty acids and decreased levels of polyunsaturated fatty acids. Additionally, hormone and glucose content analyses revealed that compared with WT lines the bnacipk9 lines showed significant differences: in bnacipk9 seeds, indoleacetic acid and abscisic acid (ABA) levels were higher; glucose and sucrose contents were higher with a higher hexose-to-sucrose ratio in bnacipk9 mid to late maturation development seeds. Furthermore, the bnacipk9 was less sensitive to glucose and ABA than the WT according to stomatal aperture regulation assays and the expression levels of genes involved in glucose and ABA regulating pathways in rapeseeds. Notably, in Arabidopsis (Arabidopsis thaliana), exogenous ABA and glucose imposed on developing seeds revealed the effects of ABA and glucose signaling on seed oil accumulation. Altogether, our results strongly suggest a role of CIPK9 in mediating the interaction between glucose flux and ABA hormone signaling to regulate seed oil metabolism in rapeseed.