<p>High-throughput phenotyping of microbial growth is crucial for understanding genotype–phenotype relationships in systems biology. Linking genetic variation to dynamic growth responses across environments remains challenging. Here, we present a time series dataset representing the growth curves of 3,909 single-gene knockout <i>Escherichia coli</i> strains grown in rich (LB) and minimal (M63) media. Using microplate assays with biological triplicates at 37 °C, we generated 23,454 OD₆₀₀ time-series trajectories (3,909 strains × 2 media × 3 replicates) recorded every 15 minutes for 24–48 hours. The dataset provides plate-background–corrected growth curves, derived growth parameters including carrying capacity (<i>K</i>) and maximal growth rate (<i>r</i>), and gene category annotations. This standardized resource facilitates comparative analyses of genotype-dependent growth dynamics between rich and poor nutritional conditions and supports methodological development for time-series processing and growth-phenotype characterization. By making the complete growth trajectories publicly available with metadata and quality indicators, we aim to enable reuse and reproducible analyses of bacterial growth dynamics across the Keio collection.</p>