Organic sulfur (S) accounts for >95 % of S in soils, making the soil S cycle particularly susceptible to changes that influence soil organic matter, such as land use change from native landscapes to agriculture. Using the Brigalow Catchment Study (BCS), a longitudinal land use change trial from semi-arid, subtropical Australia, we examined the effect of land use change on soil S composition to assess potential S availability over time. Bulk soil (0-10 cm depth) from three land uses, undisturbed (native Brigalow), cropping, and pasture, were analysed using X-ray absorption near-edge structure (XANES) spectroscopy for in situ analyses of S speciation. The concentration of total S in the bulk soil ranged 178-286 mg kg(-1) with organic S accounting for 95-99 % (176-271 mg kg(-1)). Over 40 years of land use change, there was a decrease in total soil S concentrations, being associated with a decrease in total organic carbon (TOC) and total soil nitrogen (TSN). In the pasture catchment total S decreased by 17 %, TOC by 14 %, and TSN by 29 %, whilst in the cropping catchment total S decreased by 34 %, TOC by 50 %, and TSN by 53 %. Changes in S composition resulted in a decreased average oxidation state of soil S in both the pasture and cropping catchments and an increased C-S:ester sulfate-S in the pasture catchment. This study has highlighted the need for better S management to avoid decreased S fertility and productivity through loss of total S and changes in S forms which may influence potential S availability.