Highly toxic Cr(VI) poses huge threats to human health and ecosystem. This study utilized biochar obtained from apple wood which is favorable for the formation of high C content biochar for removing Cr(VI) from aqueous media. Cr(VI) removal was highly pH-dependent with the highest Cr(VI) removal efficiency (99.9%) at pH 2.0. Fourier-transform infrared spectroscopy (FTIR) results showed that the functionalities CO and CO on biochar were likely involved in Cr(VI) treatment. Results of X-ray photoelectron spectroscopy (XPS) analysis and X-ray absorption near-edge structure (XANES) spectra indicated that the majority of Cr exhibited as the reduced Cr(III) on the biochar. Confocal micro X-ray fluorescence (μ-XRF) maps confirmed the heterogeneous distribution of Cr on biochar. The electrostatic attraction, Cr(VI) reduction, Cr(III) complexation, and ion exchange likely accounted for the principal processes of Cr(VI) removal from water. These results showed that biochar can be an effective reactive medium for remediation of Cr(VI) in an aqueous solution. This study firstly integrated the Cr(VI) removal data with XANES and confocal μ-XRF mapping to obtain a deeper understanding of Cr speciation and distribution on biochar, which was critical for identifying the key role of functional groups and Cr(VI) removal mechanisms using apple wood biochar.