Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homeostasis against external Pi variations in Arabidopsis and rice, while it is poorly understood for the identity and regulatory mechanism of VPTs in Brassica napus ( B. napus). Here, we identified two vacuolar Pi influx transporters BnA09PHT5;1b and BnCnPHT5;1b in B. napus and uncovered their necessity for cellular Pi homeostasis through functional analysis. BnPHT5;1bs are the homologs of Arabidopsis AtPHT5;1 with the similar sequence, structure, tonoplast localization, and VPT activity. BnPHT5;1b double mutants had smaller shoot growth and higher shoot cellular Pi than the wild-type B. napus, which are largely different from the report in At PHT5;1 mutant, suggesting PHT5;1-VPTs play a distinct mechanism of cellular Pi homeostasis in seedlings of B. napus and Arabidopsis. By contrast, disruption of BnPHT5;1b genes slowed vegetative growth accompanied by Pi toxicity in floral organs, reduced seed yield and impacted seed traits, agreeing with the role of AtPHT5;1 in floral Pi homeostasis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved regulatory mechanisms of BnPHT5;1bs in cellular Pi homeostasis in this plant species.