<p>Fossil fuel combustion-derived CO₂ has a lower ratio of ¹³C than the CO₂ naturally existing in the atmosphere and dilutes the overall level of ¹³C at a global scale. However, the effects of CO₂ with low ¹³C/¹²C on algae and the relevant underlying mechanisms remain elusive. In this study, the effects of ¹³C-depletion and high concentration of carbon sources (LA-HC) on carbon and nitrogen metabolism in <i>Nannochloropsis oceanica</i> (<i>N. oceanica</i>) were investigated using stable isotope techniques and transcriptomics. Results showed that LA-HC increased the growth, carbon fixation, and nitrogen fixation rates of <i>N. oceanica</i> by 21.83%, 19.35%, and 14.71%, respectively. Stable isotope analysis indicated that LA-HC significantly decreased the <i>δ</i>¹³C values of <i>N. oceanica</i>, and the algae preferentially fixed CO₂ containing ¹²C. Furthermore, LA-HC limited nitrogen isotope fractionation, and the algae were enriched in the heavier isotope ¹⁵N, indicative of higher nitrogen fixation. Fatty acid analysis indicated that LA-HC induced polyunsaturated fatty acids accumulation, especially C20:5n-3, which plays a key role in promoting photosynthetic membrane activity. In terms of amino acid content, the increase in phenylalanine, glutamine, and proline levels promoted nitrogen storage in <i>N. oceanica</i>. Transcriptomics revealed that LA-HC upregulated the genes involved in glycolysis, the tricarboxylic acid cycle (TCA cycle), and nitrogen metabolism, which increased energy metabolism and promoted fatty acid and amino acid synthesis, thereby enhancing carbon and nitrogen metabolism in <i>N. oceanica</i>. This study provides new insights into the mechanism of carbon and nitrogen metabolism in algae and is meaningful for studying the marine carbon and nitrogen cycle.</p>