The coordination bond between gold and sulfur (Au-S) has been widely studied and utilized in many fields. However, detailed investigations on the basic nature of this bond are still lacking. A gold-specific binding protein, GolB, was recently identified, providing a unique opportunity for the study of the Au-S bond at the molecular level. We probed the mechanical strength of the gold sulfur bond in GolB using single-molecule force spectroscopy. We measured the rupture force of the Au-S bond to be 165 pN, much lower than Au-S bonds measured on different gold surfaces (similar to 1000 pN). We further solved the structures of apo-GolB and Au(I) GolB complex using X-ray crystallography. These structures showed that the average Au-S bond length in GolB is much longer than the reported average value of Au-S bonds. Our results highlight the dramatic influence of the unique biological environment on the stability and strength of metal coordination bonds in proteins.