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Purpose: Herein, we examined C. necator for its production of bioplastic under mixotrophic fermentation. The mixotrophic process utilized dual carbon sources of mixed CO2 and glucose for the production of PHB. Methods: C. necator was optimized through adaptive laboratory evolution under a mixed carbon sources of CO2 and glucose. The isolated mutant was then studied for its ability to co-utilize glucose and CO2 carbon sources for growth and for PHB production. Experimental design based on central composition design was implemented to optimize PHB production under mixotrophic fermentation. Parameters effecting PHB accumulation including CO2 and glucose substrate ratio, cell dosage and aeration were studied. Results: Under optimized mixotrophic batch process, 0.22 g/L of PHB and 28% PHB content was reached from mixed carbon sources. Further CO2 and glucose co-feeding strategy optimization in mixotrophic fed-batch, PHB titer was elevated to 0.41 g/L. Conclusion: Overall, this study offered a promising alternative for CO2 valorization through the mixotrophic conversion of CO2 and glucose to PHB by C. necator, which could provide basis in future Bio-CCU technology development for climate change mitigation. [ABSTRACT FROM AUTHOR]