Artificial synthesis of glucose, the monomer of starch, from renewable resources and CO2 is a promising method for addressing food crisis and alleviating climate change. Here, the construction of a microbial biocatalyst for glucose production from renewable resources and CO2 was reported. Initially, blocking the glucose catabolic pathway via deletion of glk gene generated a glucose-producing strain of Cupriavidus necator with titers of 24.7, 47.5 and 180.1 mg/L from fructose, glycerol and CO2, respectively. Subsequently, the Entner-Doudoroff pathway and polyhydroxybutyrate biosynthesis pathway were disrupted to further increase glucose accumulation. The maximum glucose titer and yield on biomass from CO2 reached 253.3 mg/L and 91.6 mg/L/OD600, respectively. Finally, the phosphatases that mediate the dephosphorylation of phosphorylated glucose were identified. Overexpression of HAD1 and cbbY2 could enhance glucose titer by 5.5-fold when fructose was used as sole carbon source. This study demonstrates a feasible route for microbial-based synthesis of glucose from CO2.