The plastid-localized nucleotide triphosphate transporter (NTT) transports cytosolic adenosine triphosphate (ATP) into plastid to satisfy the needs of biochemistry activities in plastid. Here, we investigate the key functions of two conserved BnaNTT1 genes, BnaC06.NTT1b and BnaA07.NTT1a, in Brassica napus. Binding assays and metabolic analysis indicate that BnaNTT1 binds ATP/adenosine diphosphate (ADP), transports cytosolic ATP into chloroplast, and exchanges ADP into cytoplasm. Thylakoid structures are abnormal and plant growth is retarded in CRISPR mutants of BnaC06.NTT1b and BnaA07.NTT1a. Both BnaC06.NTT1b and BnaA07.NTT1a play important roles in the regulation of ATP/ADP homeostasis in plastid. Manipulation of BnaC06.NTT1b and BnaA07.NTT1a causes significant changes in glycolysis and membrane lipid composition, suggesting that increased ATP in plastid fuels more seed-oil accumulation. Together, this study implicates the vital role of BnaC06.NTT1b and BnaA07.NTT1a in plant metabolism and growth in B. napus.