This paper introduces some latest developments regarding the X-ray imaging methodology and applications of the X-ray imaging and biomedical application beamline (BL13W1) at Shanghai Synchrotron Radiation Facility in the past 5 years. The photon energy range of the beamline is 8–72.5 keV. Several sets of X-ray imaging detectors with different pixel sizes (0.19–24 μm) are used to realize X-ray microcomputed tomography (X-ray micro-CT) and X-ray in-line phase-contrast imaging. To satisfy the requirements of user experiments, new X-ray imaging methods and image processing techniques are developed. In vivo dynamic micro-CT experiments with living insects are performed in 0.5 s (sampling rate of 2 Hz, 2 tomograms/s) with a monochromatic beam from a wiggler source and in 40 ms (sampling rate of 25 Hz, 25 tomograms/s) with a white beam from a bending magnet source. A new X-ray imaging method known as move contrast X-ray imaging is proposed, with which blood flow and moving tissues in raw images can be distinguished according to their moving frequencies in the time domain. Furthermore, X-ray speckle-tracking imaging with twice exposures to eliminate the edge enhancement effect is developed. A high-precision quantification method is realized to measure complex three-dimensional blood vessels obtained via X-ray micro-CT. X-ray imaging methods such as three-dimensional X-ray diffraction microscopy, small-angle X-ray scattering CT, and X-ray fluorescence CT are developed, in which the X-ray micro-CT imaging method is combined with other contrast mechanisms such as diffraction, scattering, and fluorescence contrasts respectively. Moreover, an X-ray nano-CT experiment is performed with a 100 nm spatial resolution. Typical user experimental results from the fields of material science, biomedicine, paleontology, physics, chemistry, and environmental science obtained on the beamline are provided.