Chemoresistance is a major factor driving breast cancer (BC) relapse and the high rates of cancer-related deaths. Aberrant levels of glycans are closely correlated with chemoresistance. The essential functions of glycans in chemoresistance is not systematically studied. In this study, an integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics was applied to explore the dysregulation of glycogenes, glycan structures and glycoproteins in chemoresistance of breast cancer cells. In paclitaxel (PTX) resistant MCF7 cells, 19 differentially expressed N-glycan-related proteins were identified, of which MGAT4A was the most significantly down-regulated, consistent with decrease in MGAT4A expression at mRNA level in PTX treated BC cells. Glycomic analysis consistently revealed suppressed levels of multi-antennary branching structures using MALDI-TOF/TOF-MS and lectin microarray. Several target glycoproteins bearing suppressed levels of multi-antennary branching structures were identified, and ERK signaling pathway was strongly suppressed in PTX resistant MCF7 cells. Our findings demonstrated the aberrant levels of multi-antennary branching structures and their target glycoproteins on PTX resistance. Systematically integrative multi-omic analysis is expected to facilitate the discovery of the aberrant glycosyltransferases, N-glycosylation and glycoproteins in tumor progression and chemoresistance. SIGNIFICANCE: An integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics is crucial to understand the association between glycans and chemoresistance in BC. In this multi-omic analysis, we identified unique glycan-related protein, glycan and glycoprotein signatures defining PTX chemoresistance in BC. This study might provide valuable information to understand molecular mechanisms underlying chemoresistance in BC.