The misalignment of segments in installation is a common issue in the construction of TBM tunnels. This raises a question of whether misalignment affects the operation safety of a hydraulic TBM tunnel. Using a water transfer engineering project as an example, this paper built a three-dimensional finite element model composited with segment, grout layer and surrounding rock for the numerical analysis of the behavior of single-directionally misaligned segments. The crown or invert segment was separately misaligned towards to the center of segment ring in a value of 5 mm, 10 mm, 20 mm, 30 mm or 40 mm. The strength grade of the segment concrete was C50. A weaker surrounding rock composed of V-class rock was considered for the tunnel. The results indicate that the misalignment of the crown or invert segment, respectively, creates the tensile stress in the inner surface of the corresponding segment, the tensile stress will be over the limit of C50 concrete when the misalignment is over 30 mm, indicating a risk of concrete cracking. The contact surfaces of the segment ring basically remain in compression, and the locating pins between the segment rings exhibit an evident increase in tensile stress at misaligned positions. The key points that can be obtained from this study are that a special supervision is needed to ensure the accuracy of segment installation, and strengthening measures are needed for existing misaligned segments.