Locally advanced rectal cancer (LARC) exhibits heterogeneous responses to neoadjuvant chemoradiotherapy (nCRT), patients resistant to nCRT failing to benefit from to nCRT. The aim of this pilot study is to identify proteins associated with LARC nCRT-resistance. This pilot study retrospectively enrolled 4 representative nCRT-resistant (stable disease/progressive disease) and 4 nCRT-sensitive (complete response/partial response) patients, of which a total of 15 formalin-fixed paraffin-embedded tumor tissues (before and after nCRT) were subjected to data-independent acquisition mass spectrometry for proteomic analysis. Spectronaut software was employed for protein identification and quantification. Principal component analysis (PCA) was used to visualize intergroup differences in detected proteins. Differently abundant proteins in resistant patients (fold change ≥ 1.2 or ≤ 0.83 compare to sensitive patients) were selected for analysis using hierarchical clustering, cluster of orthologous groups, subcellular location, gene ontology, and pathway enrichment assay. A total of 39052 peptides and 6006 proteins were identified. PCA showed PCA clear separation among four groups: pre-treatment sensitive (B_S), pre-treatment resistant (B_R), post-treatment sensitive (P_S), and post-treatment resistant (P_R). Intergroup comparisons revealed before nCRT, 133 proteins were upregulated in the resistant group compared to the sensitive group, mainly localized in cytoplasm (33.08%), secreted (24.81%), and nucleus (24.06%), with functions enriched in cell junctions and metabolic related pathways. Post nCRT, 290 proteins were upregulated in the resistant group compared to the sensitive group, mainly localized in the nucleus (36.55%) and cytoplasm (23.10%), with functions enriched in replication, transcription, translation, and metabolic pathways. This study characterized DAP signatures of LARC with nCRT resistance, specific DAPs in resistant tumor provide references for mechanistic studies on treatment resistance, could be potential biomarker for response and therapeutic targets after further validation in larger cohorts.