Objective Performance and physiological responses of dairy calves may change by using extruded soybean meal (ESBM) instead of common soybean meal (SBM) in starter feed. The aims of the current study were 1) to determine the effect of extrusion processing of SBM on protein electrophoretic size, fourier transform infrared spectroscopy (FTIR) structures and CNCPS protein sub-fractions and 2) to determine the effect of substitution of SBM with ESBM in starter feed of Holstein heifer calves during pre and post-weaning on performance, nutrient digestibility, and blood metabolites. Methods The SBM was substituted with ESBM at the level of 0, 25, 50, 75 and 100 % (DM basis). Fifty heifer calves (initial body weight 40.3±0.63 kg) were used for the study. After birth, animals were fed colostrum for 3 days and then they were fed whole milk until weaning. Animals had free access to starter feed and water during the study. Results Extrusion of SBM decreased electrophoretic protein size and increased rapidly degradable true protein fraction, changed FTIR protein and amide II region. With increasing level of ESBM in the diet, starter intake increased quadratically during the pre-weaning period (p < 0.05) and body weight, DM intake and average daily gain increased linearly during the post-weaning and the whole study period (p < 0.05). Dry matter and CP digestibilities at week 14 and blood glucose and BHBA increased linearly in calves as the level of ESBM increased in the diet (p < 0.05). Conclusion Dairy calves performance and physiological responses were sensitive to SBM protein characteristics including electrophoretic size, FTIR structures and CNCPS protein fractions.