Soy insoluble fiber (IF), co-product of enzyme-assisted aqueous extraction process (EAEP) of soybeans, is rich in carbohydrate and protein. It can be used to enhance ethanol production in an integrated corn-soy biorefinery, which integrates components from soybean processing into corn-based ethanol processing. However, cornstarch and IF have unique carbohydrate compositions that require different treatments for optimal fermentation. The present study investigated the effect of pretreatment method [soaking in aqueous ammonia (SAA), liquid hot water (LHW), and enzymatic hydrolysis], simultaneous saccharification and co-fermentation (SSCF) with Saccharomyces cerevisiae and Escherichia coli KO11, and scaling up from bench scale (150 mL) to pilot scale (60 L) on ethanol production in IF fermentation. Untreated IF was added to integrated corn-soy fermentation and the effect of SSCF was evaluated. It was demonstrated that enzymatic hydrolysis with enzyme cocktail of pectinase, cellulase and xylanase, is the best pretreatment method to maximize ethanol production in IF fermentation with an added advantage of adding enzymes to the fermentation slurry at the SSF step. Ethanol yield almost doubled when SSCF of IF was performed with E. coli KO11 due to conversion of arabinose and xylose into ethanol. Addition of untreated IF to dry-grind corn fermentation increased ethanol production rate, but low ethanol tolerance of E. coli KO11 was a limiting factor in achieving SSCF with S. cerevisiae and E. coli KO11. Michaelis-Menten equation accurately predicted E. coli KO11 growth kinetics by Hanes-Woolf linearization.