Lesion and inactivation methods have played important roles in neuroscience studies. However, traditional techniques for creating a brain lesion are highly invasive, and control of lesion size and shape using these techniques is not easy. Here, we developed a novel method for creating a lesion on the cortical surface via 365 nm ultraviolet (UV) irradiation without breaking the dura mater. We demonstrated that 2.0 mWh UV irradiation, but not the same amount of non-UV light irradiation, induced an inverted bell-shaped lesion with neuronal loss and accumulation of glial cells. Moreover, the volume of the UV irradiation-induced lesion depended on the UV light exposure amount. We further succeeded in visualizing the lesioned site in a living animal using magnetic resonance imaging (MRI). Importantly, we also observed using an optical imaging technique that the spread of neural activation evoked by adjacent cortical stimulation disappeared only at the UV-irradiated site. In summary, UV irradiation can induce a focal brain lesion with a stable shape and size in a less invasive manner than traditional lesioning methods. This method is applicable to not only neuroscientific lesion experiments but also studies of the focal brain injury recovery process.