Palmitoleic acid (POA) can be naturally found only in few oil seeds and has significant applications in pharmaceutical industry. Recently, the isolated oleaginous yeast Scheffersomyces segobiensis DSM 27193 was identified with high content of POA in its intracellular lipid (13.80%). In this study, process optimization focused on dissolved oxygen regulation to improve POA production was conducted. Dynamic agitation was found to do significant enhancement on POA-rich lipid production than aeration regulation. Under the best condition of 1000 r·min−1 of agitation and 1 vvm of aeration, no ethanol was detected during the whole fermentation process, while a dry biomass concentration of 44.80 g·L−1 with 13.43 g·L−1 of lipid and 2.93 g·L−1 of POA was achieved. Transcription analysis revealed that the ethanol synthetic pathway was downregulated under the condition of high agitation, while the expression of the key enzymes responsible for lipid and POA accumulation were enhanced.