Abstract: The variety ‘Shitai No.1’of Pseudostellaria heterophylla was used for experimental material.Different nitrate usages were designed to study the growth and development,as well as heterophyllin B content of P.heterophylla by tissue culture method,in order to provide reference for the nitrogen dosage in the cultivation of P.heterophyllatissue culture seedlings.The results showed that the plants of P.heterophylla with no nitrogen addition had a phenotype of short plant,leaves chlorosis,less root formation and enlongation;while with nitrate addition as N resource,the plant could grow normally with higher plant height,more leaves with dark green,more roots formation.Compared with no N addition,supplying nitrate significantly increased the activities of antioxidant enzymes and N metabolism enzymes,enhanced the mineral nutrients uptake.The best growth of P.heterophylla was achieved at supplying 20mmol·L^-1 nitrate;the highest heterophyllin B content and prePhHBexpression of P.heterophylla,which were positively correlated with the N content,were both gained at supplying 10 mmol· L^-1 nitrate.This means improving N content of P.heterophylla could increase the accumulation of heterophyllin B.In conclusion,supplying 10-20mmol·L^-1 nitrate as the N resource in tissue culture of P.heterophylla,could promote the growth and development as well as the accumulation of heterophyllin B.