This study has demonstrated the feasibility of substituting 50 g/100 g of wheat flour (WF, with a very high gluten content and intermediate amylopectin percentage in starch) with highland barley powder (HBP, with a very high TDF content and amylopectin percentage in starch) or mung bean flour (MBF, with a very high crude protein) for chiffon cake making. Either substitution modified largely batter properties (batter specific gravity and viscosity: WF + HBP > WF + MBF > WF alone), and cake crumb structure setting during baking via affecting starch gelatinization, protein coagulation/aggregation, migration, and coalescence of both fat particles and gas cells. The amylopectin percentage in starch for WF, MBF, and HBP was strongly positively correlated with the flow behavior index (n) of corresponding cake batters (r = 0.892). The cell-to-total area ratio of cakes (WF alone ≈ WF + MBF > WF + HBP) was strongly positively correlated with the enthalpy of heat absorption (△H) of the ingredient powder (r = − 0.678), while strongly negatively correlated with the air-holding capacity, consistency coefficient (K), and n of cake batters (r = − 0.769, − 0.941, and − 0.628, respectively). The cell density/cell-to-total area ratio of cakes (WF + HBP > WF alone > WF + MBF) was strongly positively correlated with the K (r = 0.749) and n (r = 0.880) values of cake batters. The cake with WF + HBP had the greatest pore distribution and highest uniformity. The cake with WF + MBF was the hardest, gummiest, and chewiest and resembled that with WF alone in crumb color with the latter having the lowest specific volume.