Poly-3-hydroxybutyrate (PHB) can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol (0.1%) was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages: a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients (ammonium, nitrate, phosphorus, copper, iron (III), magnesium or ethylenediamine tetraacetate (EDTA)) in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle (TCA). It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% (w/w) from the initial yield of 12% (w/w) after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5 ×106Da.