Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy (AFM) experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (~1 kBT). Applying pulling forces of only 5 pN we maintain gpW in quasiequilibrium near its mechanical unfolding midpoint, and detect how it interconverts stochastically between the folded and an extended state. This binary pattern indicates that, under an external force, gpW (un)folds over a significant free energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single molecule force spectroscopy.Deciphering the mechanisms by which proteins fold has long been one of the central problems in molecular biophysics 1,2 . This quest has proved challenging because most single domain proteins fold slowly via a two-state (i.e. all or none) process 3 , and atomistic simulations could only access very short timescales 4 . In this context, downhill folding attracted particular attention with the promise of unveiling details of folding energy landscapes that are hidden in two state folding 5 .Downhill folding proteins do not cross significant free energy barriers and thus exhibit limited cooperativity 6 and are amongst the fastest to fold and unfold 7 . Their s folding times have been instrumental in bridging the time scale gap between experiment and atomistic molecular dynamics (MD) simulations 7-11 . The minimal cooperativity of downhill folding has led to methods that distil mechanistic information from conventional ensemble experiments, such as monitoring how thermal denaturation depends on the structural probe 12 , analyzing heat capacity thermograms in terms of low-dimensional free energy surfaces 13 , or estimating free energy barriers to folding from the curvature of the Eyring plot 14 .Whereas many fast folding proteins share common structural features like their small size (typically, less than 45 residues) or primarily helical secondary structure (with the exception of the very small WW domains), the protein gpW is an outlier to these general trends 15 . gpW has 65