Environmental stresses such as nitrate deprivation and high light are effective at increasing lipid content in microalgae, but they can also slow down and even stop growth. In this study, the phytohormones methyl jasmonate, salicylic acid, gibberellin, abscisic acid, and ethephon were introduced to cultures of the oleaginous marine diatom Phaeodactylum tricornutum in an attempt to increase growth and lipid production. Single-factor experiments showed that the influences of some of the phytohormones were closely related to their concentrations. Methyl jasmonate, abscisic acid, and salicylic acid promoted P. tricornutum growth and lipid accumulation at certain concentrations. The differing effects of the three phytohormones on P. tricornutum may be related to the respective phytohormone's responsive cis-regulatory elements in the upstream regions of the triacylglycerol (TAG) synthesis genes. Methyl jasmonate, abscisic acid, and salicylic acid were further studied in response surface experiments, through which a 141% increase in TAG production was attained for 10-L cultures of P. tricornutum grown under optimal conditions. This study suggests that some phytohormones can promote P. tricornutum lipid accumulation without hindering growth. It also provides another strategy for improving the production of microalgae for use as biodiesel.