BACKGROUND High precision radiotherapy with small irradiator size has potential in many treatment applications involving small shallow targets, with small animal radio-neuromodulation as an intriguing example. A focused kV technique based on novel usage of polycapillary x-ray lenses can focus x-ray beams to <0.2 mm in diameter, which is ideal for such uses. OBJECTIVE Such an application also requires high resolution CT images for treatment planning and setup. In this work, we demonstrate the feasibility of using a virtual focal spot generated with an x-ray lens to perform high-resolution CBCT acquisition. METHODS The experiment with x-ray lens was set up on an x-ray tabletop system to generate a virtual focal spot. The flood field images with and without the x-ray lens were first compared. A pinhole image was acquired for the virtual focal spot and compared with the one acquired with the conventional focal spot without the lens. The planar imaging resolution with and without the lens were evaluated using a line pair resolution phantom. The spatial resolution of the two settings were estimated by reconstructing a 0.15-mm wire phantom and comparing its full width half maximum (FWHM). A CBCT scan of a rodent head was also acquired to further demonstrate the improved resolution using the x-ray lens. RESULTS The proposed imaging setup with x-ray lens had a limited exposure area of 5 cm by 5 cm on the detector, which was suitable for guiding radio-neuromodulation to a small target in rodent brain. Compared to conventional imaging acquisition with a measured x-ray focal spot of 0.395 mm FWHM, the virtual focal spot size was measured at 0.175 mm. The reduction in focal spot size with lens leads to an almost doubled planar imaging resolution and a 26% enhancement in 3D spatial resolution. A realistic CBCT acquisition of a rodent head mimicked the imaging acquisition step for radio-neuromodulation and further showed the improved visualization for fine structures. CONCLUSIONS This work demonstrated that the focused kV x-ray technique was capable of generating small focal spot size of <0.2 mm, which substantially improved x-ray imaging resolution for small animal imaging.