Vancomycin resistance of Gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused reduced susceptibility to vancomycin in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to β-lactams and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had (i) a decreased amount of lipoteichoic acid, (ii) decreased biofilm formation, and (iii) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose. These phenotypes and vancomycin tolerance were abolished by knockout of ykcC, a gene in the same operon with ykcB probably involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin tolerance in both the parent strain and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to reduced susceptibility to vancomycin, decreased biofilm formation, and increased pathogenicity to silkworms. IMPORTANCE Although vancomycin is effective against Gram-positive bacteria, vancomycin-resistant bacteria are a major public health concern. While the vancomycin-resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well studied, they remain unclear in other Gram-positive bacteria. In the present study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin tolerance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in Gram-positive bacteria.