Survivin, as an apoptosis suppressor, exists as a homodimer interfacing at the N-terminal portion (residues 6-13) of its baculovirus IAP repeat (BIR) domain and a linker segment (residues 89-102). Here we expressed full-length human Survivin (SurF) and a series of its mutants, SurΔN7, SurΔN13, and SurΔN18 with significant truncations of the N-terminus, all of which could still dimerize in solution. Single-molecule force spectroscopy (SMFS) was used to quantitate the unbinding forces of full-length and the mutant homodimers and revealed that the N-terminal residues up to Arg18 were not essential for dimerization. Meanwhile, the binding of SurΔN7 to Smac/DIABLO determined by ELISA was as efficient as the wild-type, but that of SurΔN13 was significantly reduced, and that of SurΔN18 was completely lost. Together, these findings provide direct evidence that the N-terminal sequence of Survivin is not critical for dimer formation but may contribute to correct folding and function of BIR.