To complete the explanation on the mechanism of the Poly-3-hydroxybutyrate (PHB) synthesis intracellular in methanotrophs, the role of nicotinamide adenine dinucleotide (NADH) in the PHB synthesis mechanism in Methylosinus trichosporium IMV 3011, which can utilize methane and methanol as source of carbon and energy to synthesize PHB, was studied. The study carried on by using carbon monoxide (CO) as NADH consumption substrate or using sodium formate as NADH production substrate. The results showed that NADH influenced the synthesis and accumulation of PHB intracellular to some extent. Especially, the yield of PHB would decrease to 13.5% (compared to normal yield of 21.3%) under NADH deficiency condition caused by CO at content of 20%. During the flask fermetation process of Methylosinus trichosporium IMV 3011, adding CO with certain centent (10%, v/v) or at right time (72h) into gas mixture would keep the NADH intrcelluar at appropriate concentration, which will be avialable for cell growth and PHB accumulation. The findings on the NADH will greatly contribute to the less expensive production of PHB by methanotrophs and promote the application in this field.