Cytosolic glutamine synthetase (GS1) plays a central role in nitrogen (N) metabolism. The importance of GS1 in N remobilization during reproductive growth has been reported in cereal species but attempts to improve N utilization efficiency (NUE) by overexpressing GS1 have yielded inconsistent results. Here, we demonstrate that transformation of barley (Hordeum vulgare L.) plants using a cisgenic strategy to express an extra copy of native HvGS1-1 lead to increased HvGS1.1 expression and GS1 enzyme activity. GS1 overexpressing lines exhibited higher grain yields and NUE than wild-type plants when grown under three different N supplies and two levels of atmospheric CO₂. In contrast with the wild-type, the grain protein concentration in the GS1 overexpressing lines did not decline when plants were exposed to elevated (800–900 μL/L) atmospheric CO₂. We conclude that an increase in GS1 activity obtained through cisgenic overexpression of HvGS1-1 can improve grain yield and NUE in barley. The extra capacity for N assimilation obtained by GS1 overexpression may also provide a means to prevent declining grain protein levels under elevated atmospheric CO₂.