Methane is considered the carbon source with the highest potential in industrial biotechnology because of its abundance and sustainability. The biological conversion of methane into chemicals or fuels can not only reduce greenhouse gas emissions, but can also substitute edible substrates used in biorefineries. Methanotrophs that can utilize methane as the sole energy and carbon source play a significant role in the ecology of methane. Studies on metabolically-engineered methanotrophs have attracted extensive attention in recent years. In this chapter, the approaches and strategies of methanotrophic cell factory construction are summarized based on synthetic biology tools, systematic manipulation, metabolic modeling, and carbon flux enhancement. Finally, the challenges and opportunities for methane bioconversion by methanotrophs are discussed based on industrial applications.