Highland barley is the main food crop in high altitude cold region of Tibet. Effects of water deficit levels at different growth stages on the water use efficiency and yield of spring highland barley (Hordeum vulgare) were studied by barrel cultivated experiments. Experimental treatments included full irrigation treatment (two controlled lower limits on soil moisture content and straw mulching) as well as four water deficit levels (mild, moderate, severe and extreme deficits) in the whole growth period and five different growth stages, respectively, and the total treatments were 27. Experimental results showed that the harvest index, grain yield and water use efficiency of spring highland barley in the treatment with the water controlled lower limit of 75% of field capacity (FC) were greater than those of 80% FC treatment, and straw mulching treatment resulted in the largest grain yield and crop water use efficiency in all experimental treatments under full irrigation condition. Under the condition of various water deficit levels in the whole growth period, the grain yield of spring highland barley was smaller than that of full irrigation treatment, and it was decreased significantly with the increase of water deficit level. Water deficit from mild to severe levels obtained larger harvest index and crop water use efficiency, but extreme deficiency on water supply resulted in the lowest grain yield, harvest index and crop water use efficiency under various water deficit treatments in the whole growth period. The grain yield and crop water use efficiency under water deficit treatments at different growth stages of spring highland barley were basically decreased with the increase of water deficit level except the treatment with water deficit at ripening stage, and the effect of water deficit at jointing, tillering and filling stages on grain yield was greater than that at other growth stages. Straw mulching or water deficit with mild to severe levels in the whole growth period of spring highland barley can improve crop water use efficiency, and their effects in practice are deserved to be paid more attentions.