Knotted polypeptide chain are found to be one of the most surprising topological features in some proteins. The topological difficulty of the folding of knotted proteins has become a challenging problem. It was reported that a structure of slipknot could serve as an important intermediate state during the folding of knotted proteins. Here we use single molecule force spectroscopy (SMFS) as well as steered molecular dynamics (SMD) simulations to transform a slipknot protein AFV3-109 into a tightened trefoil knot. Our results show that by pulling on the N-terminus and the threaded loop of AFV3-109, the protein can be unfolded via multiple pathways and the slipknot would be transformed into a tightened trefoil knot with ∼13 amino acid residues involved because the polypeptide chain is apparently shortened by ∼4.7 nm. The SMD simulations agree with our experiments and provide detailed molecular mechanism of mechanical unfolding and knot tightening of AFV3-109. Interactions between shearing β-strands on threaded loop and knotting loop provide high mechanical resistance in the process of forming the trefoil knot, i.e., pulling threaded loop through knotting loop.