Glycine is an amino acid that functions as both a carbon and nitrogen source. Glycine mixotrophy enhances microalgal biomass, reduces dependency on light, and optimizes carbon and nitrogen allocation, thereby facilitating the adaptation of microalgae to industrial production environments. This study investigated the physiological and biochemical parameters, as well as gene expression profiles, of Isochrysis zhanjiangensis under varying glycine concentrations to elucidate the effects of glycine on fucoxanthin content and yield, along with its underlying mechanisms. At the physiological and biochemical level, fucoxanthin yield peaked at 4.74 mg/L under an 8 g/L glycine concentration, a 6.36-fold increase compared to the control group. Biomass reached its maximum of 9.38 × 106 cells·mL-1 at a 2 g/L glycine concentration, reflecting a 62.41% increase over the control. Chlorophyll content and fluorescence parameters revealed that glycine treatment increased chlorophyll levels, enhanced the photosynthetic activity of photosystem II (PSII), and mitigated photoinhibition. At the gene expression level, transcriptome analysis indicated that glycine significantly upregulated the expression of genes encoding enzymes in the Calvin cycle, potentially enhancing carbon fixation and providing energy for algal cell proliferation. A portion of the carbon flux was redirected toward fatty acid storage. Additionally, the fucoxanthin biosynthesis pathway gene ZEP1 exhibited elevated expression, collectively contributing to the observed increase in fucoxanthin yield.