Since protein aggregation and formation of a continuous protein matrix in rye dough is very limited, an enzyme-induced protein aggregation method to improve the baking properties was investigated. The effects of microbial transglutaminase (TG) on the properties of rye dough were studied by rheological tests, confocal laser scanning microscopy (CSLM), standard-scale baking tests and crumb texture profile analysis. Addition of TG in the range of 0–4000 Ukg −1 rye flour modified the rheological properties of rye flour dough, resulting in a progressive increase of the complex shear modulus (| G∗|) and in a decrease of the loss factor (tan δ) due to protein cross-linking or aggregation. CLSM image analysis illustrated a TG-induced increase of the size of rye protein complexes. Standard baking tests showed positive effects on loaf volume and crumb texture of rye bread with TG applied up to 500 Ukg −1 rye flour. Higher levels of TG (500 U ≤ TG ≤ 4000 U) had detrimental effects on loaf volume. Increasing TG concentration resulted in an increase of crumb springiness and hardness. In conclusion, the results of this work demonstrated that TG can be used to improve the bread making performance of rye dough by creating a continuous protein network.