Terahertz (THz) reflective imaging is applied to the stratigraphic and subsurface investigation of oil paintings, with a focus on the mid-20th century Italian painting, ‘After Fishing’, by Ausonio Tanda. THz frequency-wavelet domain deconvolution, which is an enhanced deconvolution technique combining frequency-domain filtering and stationary wavelet shrinkage, is utilized to resolve the optically thin paint layers or brush strokes. Based on the deconvolved terahertz data, the stratigraphy of the painting including the paint layers is reconstructed and subsurface features are clearly revealed. Specifically, THz C-scans and B-scans are analyzed based on different types of deconvolved signals to investigate the subsurface features of the painting, including the identification of regions with more than one paint layer, the refractive-index difference between paint layers, and the distribution of the paint-layer thickness. In addition, THz images are compared with X-ray images. The THz image of the thickness distribution of the paint exhibits a high degree of correlation with the X-ray transmission image, but THz images also reveal defects in the paperboard that cannot be identified in the X-ray image. Therefore, our results demonstrate that THz imaging can be considered as an effective tool for the stratigraphic and subsurface investigation of art paintings. They also open up the way for the use of non-ionizing THz imaging as a potential substitute for ionizing X-ray analysis in nondestructive evaluation of art paintings.