Southern Ocean phytoplankton experience low temperature, low irradiance, and strong seasonal variability. Changes in irradiance and temperature can directly affect primary productivity and the consequent carbon and nitrogen cycling. However, physiological and community-level functional responses of natural phytoplankton communities in different Southern Ocean sectors remain poorly understood. In this study, a series of shipboard batch incubation experiments were performed using natural phytoplankton communities collected from three regions with different environmental and nutrient conditions: the Antarctic Peninsula, the Cosmonaut Sea, and the Prydz Bay. A 22 factorial experimental treatments were set at two temperature levels (0°C and 4°C) and two irradiance levels (medium light: ~50 μmol photons m⁻² s⁻¹ and low light: ~10 μmol photons m⁻² s⁻¹). Physiological, relative stable-isotope signals, and molecular (18S rRNA and transcriptomic) measurements were used to evaluate region-specific responses. Distinct patterns in physiological traits and community composition were observed across the three sampling regions. In the Antarctic Peninsula, particulate organic carbon slightly increased under low light, with higher C/N ratios. In the Cosmonaut Sea, chlorophyll a increased at 4°C, and C/N ratios were slightly higher under low light. In the Prydz Bay, low light reduced particulate organic carbon, atom% ¹³C, and C/N ratios. 18S rRNA analyses showed that Fragilariopsis declined under low light, whereas dinoflagellates and green algae were more abundant in the Cosmonaut Sea. In the Prydz Bay, community structure was strongly temperature-dependent, with Mediophyceae dominating at 0°C and Fragilariopsis increasing at 4°C. Metatranscriptomic analyses indicated that low light generally upregulated photosystem, electron transport, and ATP synthesis genes, compensating for reduced irradiance. In the Prydz Bay, the low-light enhancement of nitrogen metabolism, together with increased particulate nitrogen, suggested altered carbon–nitrogen allocation. Phytoplankton responses to warming varied among regions, with increased photosystem functions at 4°C in the Cosmonaut Sea, enhanced photosystem and Calvin cycle genes in the Antarctic Peninsula, and declines in most photosynthetic and metabolic functions in the Prydz Bay, indicating constrained responses to warming. Collectively, these results demonstrate that responses of Southern Ocean phytoplankton communities to light and temperature changes are shaped by regional nutrient availability and community composition. Our findings underscore the importance of considering regional differences in community structure and carbon–nitrogen allocation strategies when assessing the responses of Southern Ocean phytoplankton to future environmental change.