Single-crystal Co3O4 nanoparticles are produced via a novel lysine-assisted hydrothermal process. When used as anode materials for lithium-ion batteries, a heat-treatment process is first introduced to decrease the initial irreversible loss and enhance the cyclability of Co3O4 nanoparticle-based electrodes using a polyvinylidene fluoride (PVDF) binder. Heat-treated electrodes exhibit improved lithium storage properties relative to those that are unheated. In particular, Co3O4 electrodes heated at 200 °C have the highest capacity and best reversibility: 1000 mA h g−1 with 95.2% capacity retention after 170 cycles at a current density of 100 mA g−1. Even when cycled at a high rate of 1000 mA g−1, a reversible capacity up to 600 mA h g−1 can still be maintained after 500 cycles. These improvements are explained based on the results from thermal analysis, transmission electron microscopy, scanning electron microscopy, nanoscratch tests, and electrochemical impedance spectroscopy measurements. Heat treatment not only improves binder distribution and adhesion to both Co3O4 particles and the substrate but also ensures high interfacial conductivity and keeps the active material particles and carbon black electrically connected, thereby leading to superior electrochemical performance. The results suggest that the heat-treated Co3O4 electrode may be a promising anode for next-generation lithium-ion batteries.