PROMALS3D is a tool designed to improve the accuracy of multiple sequence alignments (MSAs) by incorporating 3D structural information. The tool identifies homologs with known 3D structures, derives structural constraints through structure-based alignments, and combines these with sequence constraints to construct a consensus alignment. This approach leverages the slower evolutionary rate of protein structures compared to sequences, enhancing alignment quality. PROMALS3D can handle multiple input structures and outperforms existing methods in both reference-dependent and reference-independent evaluations. The tool is available as a web server and has been tested on benchmark databases such as SABmark and PREFAB, demonstrating significant improvements in alignment quality, especially for distantly related proteins.PROMALS3D is a tool designed to improve the accuracy of multiple sequence alignments (MSAs) by incorporating 3D structural information. The tool identifies homologs with known 3D structures, derives structural constraints through structure-based alignments, and combines these with sequence constraints to construct a consensus alignment. This approach leverages the slower evolutionary rate of protein structures compared to sequences, enhancing alignment quality. PROMALS3D can handle multiple input structures and outperforms existing methods in both reference-dependent and reference-independent evaluations. The tool is available as a web server and has been tested on benchmark databases such as SABmark and PREFAB, demonstrating significant improvements in alignment quality, especially for distantly related proteins.