In this study, we investigated the distribution and chemical speciation of Se in Se-rich soil by using micro-focused X-ray absorption near-edge structure (mu-XANES) spectroscopy coupling with X-ray fluorescence (mu-XRF) mapping. The microscale distribution showed that Se is heterogeneously distributed in the soil from seleniferous areas in Enshi, China. Se K-edge mu-XANES analysis suggested that Se is mainly present as Se(IV), organic Se(-II) or Se(0) species in Se-rich agricultural soil. The findings from this study would help improve the understanding of the fate, mobility, bioavailability, and biogeochemical cycling of Se in the seleniferous soil environment.