Proteins normally fold in crowded cellular environments. Here we use a set of Desulfovibrio desulfuricans apoflavodoxin variants to assess-with residue-specific resolution-how apoflavodoxin's folding landscape is tuned by macromolecular crowding. We find that, under crowded conditions, initial topological frustration is reduced, subsequent folding requires less ordering in the transition state, and beta-strand 1 becomes more important in guiding the process. We propose that conditions more closely mimicking the cellular environment make the ensemble of unfolded conformations less expanded, resulting in a folding funnel that is smoother and narrower.