Although carbohydrates are a principal component of the human diet, the mechanism of the final stages of starch digestion is not fully understood. One approach to treating metabolic diseases such as type II diabetes, obesity, and congenital sucrase isomaltase deficiency is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals: the release of free glucose. MGAM and SI consist of two catalytic subunits: N-terminal and C-terminal, with overlapping, but variant substrate specificities. The objective of this thesis is to increase the understanding of the differential substrate specificity seen in the catalytic subunits of SI and MGAM. Through inhibitor studies, the structural and biochemical differences between the enzymatic subunits are explored, illustrating that each individual catalytic subunit can be selectively inhibited. In Chapter 3, homology models of ctSI and ctMGAM-N20 are presented, giving insight into the residues hypothesized to impact substrate specificity, enhancing our understanding of the functionality of these enzymatic subunits and overlapping substrate specificity. The structural implications of mutations seen in ntSI in CSID patients and the potential functional and structural implications are discussed in Chapter 4 in addition to the prevalence of SNPs in the SI gene in different populations. The mammalian α-glucosidases are compared to the 3 Å structure of CfXyl31, a Family 31 glycoside hydrolase from Cellulomonas fimi. Comparison to Family 31 glycoside hydrolases of known structure gives rise to possible mutations proposed to mimic ntMGAM α-glucosidase activity. Through inhibitor studies, homology models, examining mutations found in disease states such as congenital sucrase isomaltase deficiency, and investigating a bacterial family 31 glycoside hydrolase from Cellulomonas fimi, the active site characteristics and substrate specificities of SI and MGAM are better understood.