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The implementation of rotational tillage with straw mulching during a fallow period seems to be an effective management strategy to help store water for spring-sown crop. A site-specific field study was conducted according to rainfall conditions to determine the effect of rotational tillage on soil water regime, water use efficiency and grain yield in semi-arid region on the loess plateau of China. Six tillage practices were tested: NT/ST rotation (no-tillage was applied in the first year and rotated with sub-soiling in the second year), ST/CT rotation (sub-soiling was applied in the first year and rotated with conventional tillage in the second year), CT/NT rotation (conventional tillage was applied in the first year and rotated with no-tillage in the second year), NT (no-tillage was applied every year), ST (sub-soiling was applied every year), CT(conventional tillage was applied every year). The results showed that the rotational tillage increase average SWS (soil water at sowing) by 5.2 mm in dry years, 0.8 mm in normal years, and 13.2 mm in humid years when compared to CT. Soil water depletion was consistent with rainfall totals, and the lowest depletion value recorded in the NT/ST and followed by NT treatment. The grain yield was positively related with rainfall, and average grain yields for three rainfall conditions were ranked as NT/ST > CT/NT > ST/CT > ST > NT = CT, while the soil water use efficiency (WUE) was ranked in the order of NT/ST, > CT/NT > ST, ST/CT > NT > CT. Grain yields of rotational tillage NT/ST, ST/CT and CT/NT are higher than the yield of NT by 6.5%-12.0%, higher than the yield of ST by 1.7%-7.0%, and 7.6%-13.2% higher than CT, respectively. Hence, rotational tillage could improve soil water storage, thus significantly increasing crop grain yield and water use efficiency. The method could have important applications in semi-arid areas.