Microalgae are being increasingly recognized as a promising solution for the search of sustainable energy resources and it has been successfully used as a feedstock for the production of liquid and gaseous biofuels. Photoautotrophic cultivation of microalgae in open ponds by utilizing sunlight and atmospheric carbon dioxide is commonly known as the most economic means for cost effective cultivation of microalgal biomass production, but is limited by optimal supply of sunlight and carbon dioxide, night biomass loss, low biomass productivity and contamination problems. Microalgae are capable of utilizing organic carbon sources, grow and metabolize in the absence of sunlight/dark conditions. Heterotrophic cultivation overcomes the light supply predicament of photoautotrophy, and in the present scenario it can be easily adapted for commercial scale production of microalgal biomass. In addition, heterotrophic cultivation is metabolically favorable for higher lipid accumulation and would be beneficial in biodiesel production. Current commercial production of polyunsaturated fatty acids are being dominated by heterotrophic marine algae. This chapter discusses in detail the heterotrophic metabolism of microalgae, the factors affecting heterotrophic cultivation and the commercial products of interest that can be obtained. The future perspectives for heterotrophic cultivation as a potential solution for obtaining large scale microalgal biomass is presented.