Microalgae have emerged as promising candidates for the sustainable production of lipids and valuable fatty acids. However, the stress strategies commonly used to enhance lipid accumulation often limit biomass accumulation, posing a significant challenge to maximizing lipid yields. Here, we adopted a two-stage cultivation strategy by separating the biomass accumulating phase at optimum temperature (Phase 1) from the lipid accumulating phase at lower temperatures (Phase 2) for Vischeria sp. WL1. We found that low temperatures at Phase 2 could rapidly induce lipid accumulation within 12 h, reaching the highest content of 61% on day 6 (2 days after transition to Phase 2). The unexpected increase in cellular chlorophyll a content at 9 degrees C might have facilitated enhanced light energy absorption for lipid accumulation. Our analysis revealed a selective decrease in saturated fatty acids, accompanied by a rise in monounsaturated fatty acids, while polyunsaturated fatty acids remained unchanged under 9 degrees C. Furthermore, fatty acids generally exhibited two distinct patterns at 9 degrees C after the transition to Phase 2, either increasing or decreasing. Notably, we discovered that the induction of fatty acid accumulation was governed by precise temperature thresholds, with 9 degrees C showing an induction effect, whereas 15 degrees C did not. Overall, our study provides practical insights into biomass accumulation, oil production, and selective fatty acid biosynthesis through a temperature-dependent two-stage cultivation strategy.