Microalgae can accumulate substantial amounts of triacylglycerol, the primary feedstock for biodiesel production, under different stress conditions. In this study, exposure to alkaline pH (pH 10) using carbonate-bicarbonate buffer, nitrogen starvation, and both were applied to induce triacylglycerol accumulation in Auxenochlorella protothecoides KP7. In addition to triacylglycerol accumulation, growth, biochemical composition, and fatty acid profiles were examined in detail and compared. Our results illustrated that the combination of nitrogen starvation and alkaline pH led to significant increases in triacylglycerol and starch contents and drastic decreases in chlorophyll and protein contents compared with their control levels. Triacylglycerol levels, the triacylglycerol/total lipid ratio, and lipid productivities were significantly increased after 7 days of cultivation under the combined stress compared with the effects of nitrogen starvation alone. After prolonged cultivation under the combined stress, triacylglycerol content reached up to 25.01% of the dry cell weight and constituted up to 81.25% of total lipids. Under the combined stress, the major fatty acids in the total lipid of A. protothecoides KP7 were C18:1 (57.5%), C18:2 (19.5%), C16:0 (12.5%), and C18:3 (5.6%), and the percentages of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids represented 16.3, 58.4, and 25.1% of the total fatty acids, respectively. Furthermore, the combined stress was associated with the lowest iodine value (97.64 g I-2 (100 g)(-1)), highest oxidative stability (7.29 h), and highest cetane number (55.26) among the examined conditions. The use of nitrogen-free medium containing carbonate-bicarbonate buffer for cultivation appears promising for enhancing triacylglycerol production in A. protothecoides.