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In the last 20 years, due to a significant reduction in industrial emissions and changes in the assortment of available fertilizers, a progressive sulphur deficiency has been observed in the soils of Poland and other parts of the world. This creates the need to supplement NPK fertilizers with sulphur and to conduct research on the optimization of its use as a fertilizer component. A three-year field study was carried out on Haplic Luvisols with low content of sulphur available to plants. The aim of the study was to assess the effect of sulphur-containing fertilizers (ammonium sulphate, potassium sulphate and Wigor S) applied at rates of 20 and 40 kg S/ha on the yield of grain, straw and protein of spring barley. The study showed that application of sulphur, irrespective of its form, generally significantly increased the yield of spring barley grain and straw as compared to the control. However, no significant differences were found in the effects of the fertilizers on yield depending on either the fertilizer type or application rate. Protein content in the grain of the plants fertilized with sulphate sulphur was on average about 6% higher than in the grain of non-fertilized plants. As a result of the beneficial effect of sulphur on the yield of spring barley grain and on its protein content, the protein yield increased. The highest increases were obtained following application of sulphate fertilizers, especially at a 40 kg S/ha. The research suggests that irrespective of the form used, sulphur fertilization in the conditions of deficiency of this nutrient in the soil, with relatively high temperatures and high soil moisture, significantly affects the yield of spring barley grain and straw and the accumulation of protein in the grain.