AbstractIntracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an ‘‐omics’ level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen‐limited medium and performed a comparative and semi‐quantitative proteomic analysis of cell samples obtained thereafter by a 2D‐LC–MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi‐quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering. Copyright © 2009 John Wiley & Sons, Ltd.