Lipopolysaccharide (LPS), which consists of a lipid A anchor, a core oligosaccharide, and a highly variable O-antigen, plays an important role in maintaining membrane integrity and preventing harmful extracellular molecules from penetrating the membrane of Gram-negative bacteria. Although the roles of lipid A and O-antigen have been extensively studied, the role of the core oligosaccharide in envelope stress responses and antibiotic resistance remains relatively poorly understood. In this study, we analyzed the roles of ten enzymes involved in core biosynthesis and modification in envelope stress responses and antibiotic resistance. Our systematic analyses demonstrated that biosynthesis and modification of heptoses present in the inner core of LPS are important for resistance to bile salts and antibiotics. Notably, we found that phosphorylation of the first heptose in the inner core had a role distinct from that of phosphorylation of the second heptose. Defects in heptose biosynthesis resulted in increased membrane permeability. Finally, we found that SpeA/SpeB-dependent putrescine biosynthesis may be partially associated with the sensitivity of the heptose biosynthesis-defective mutant to clindamycin and erythromycin. Taken together, our results demonstrate that the biosynthesis and modification of heptoses in the inner core of LPS are strongly associated with the envelope stress response and antibiotic resistance.