Cyclin-dependent kinase 6 (CDK6) plays a central role in G1–S phase cell cycle progression and is frequently dysregulated in various cancers, making it an established therapeutic target. Although selective CDK4/6 inhibitors are clinically available, exploration of natural compounds targeting CDK6 remains limited. The present study aimed to investigate the binding mechanism and inhibitory potential of limonene against CDK6 using integrated computational and experimental approaches. Recombinant CDK6 was cloned, expressed, and purified, followed by molecular docking, fluorescence spectroscopy, and kinase inhibition assay. Docking analysis revealed a binding free energy of -6.3 kcal mol−1 with a calculated pKi of 4.62 and ligand efficiency of 0.63 kcal mol−1 per non-hydrogen atom. Fluorescence quenching studies demonstrated strong binding affinity (K = 4.1 × 107 M−1), while enzymatic assays confirmed dose-dependent suppression of CDK6 activity. Collectively, these findings indicate that limonene directly interacts with and functionally inhibits CDK6, highlighting its potential as a natural scaffold for the development of CDK6-targeted anticancer therapeutics.