Biodiesel from microalgae provides a promising alternative for biofuel production. Microalgae can be produced under three major cultivation modes, namely photoautotrophic cultivation, heterotrophic cultivation, and mixotrophic cultivation. Potentials and practices of biodiesel production from microalgae have been demonstrated mostly focusing on photoautotrophic cultivation; mixotrophic cultivation of microalgae for biodiesel production has rarely been reviewed. This paper summarizes the mechanisms and virtues of mixotrophic microalgae cultivation through comparison with other major cultivation modes. Influencing factors of microalgal biodiesel production under mixotrophic cultivation are presented, development of combining microalgal biodiesel production with wastewater treatment is especially reviewed, and bottlenecks and strategies for future commercial production are also identified.