Phaeodactylum tricornutum is abundant in eicosapentaenoic acid and fucoxanthin. Heterotrophically modifying P. tricornutum to construct a production system by fermentation has attracted increasing interest. Here, glucose used heterotrophic transformant was obtained by expressing of the human Gult1 gene in P. tricornutum. It was found that glucose metabolism was mainly dependent on the PPP pathway, while the EMP pathway was regularly embolized, and excessive G6P accumulation inhibits the heterotrophic growth of the Gult1 transformant. 6-phosphofructokinase gene (Pfk), glucose-6-phosphate dehydrogenase gene (G6pd), and 6-phosphogluconolactonase gene (Pgl) were expressed in the Gult1 transformant respectively, to enhance the EMP (Embden-Meyerhof-Parnas) and PPP (Pentose Phosphate Pathway) pathways for accelerating G6P metabolic rate. The Pfks expressed transformants have 14–33 % biomass higher than Gult1 transformant under heterotrophy and 4 times higher than autotrophy. The G6pds and Pgls expressed transformants have 40 % biomass higher than Gult1 transformant under heterotrophy and 4.5 times higher than autotrophy. These findings evidenced that heterotrophic modification and further optimizing global glucose metabolism are feasibly promising to construct a cell factory for P. tricornutum industrial fermentation.