This paper investigated Mg2+ enhancement of biomass production through regulating the generation and use of energy in Rubrivivax gelatinosus wastewater treatment. Results showed that proper Mg2+ dosage range was 1.5-15 mg/L. With optimal Mg2+ dosage (10 mg/L), biomass production (5010 mg/L) was improved by 60%. Both protein and chemical oxygen demand (COD) removals reached above 90%. Biomass yield improved by 38%. Hydraulic retention time was shortened by 25%. Mechanism analysis indicated that as activator, Mg2+ promoted specifically isocitrate dehydrogenase (IDH) and Ca2+ / Mg2+ -ATPase activities in energy metabolism, and then improved the generation of adenosine triphosphate (ATP) and the use of ATP. This enhanced the secretion and activity of protease, protein and COD removals, and then led to more biomass production. With 10 mg/L Mg2+, IDH and Ca2+ / Mg2+ -ATPase activities, ATP production, protease activity were improved by 43.8%, 40.6%, 39.4% and 46.5%, respectively.