Background <p>Capsaicin, phytochemical component in red hot chili peppers, has previously been demonstrated to exhibit antitumor effect in various cancer type. However, the deep biological function and molecular mechanism of capsaicin was still uncertain.</p> Methods <p>This study applied cell viability assay, apoptosis assay, Transwell assay, immunofluorescence, qPCR, western blot assay and high-throughput RNA sequencing to explore the effect of capsaicin on gene expression and chemotherapy sensitization in gastric cancer cells.</p> Results <p>Capsaicin could significantly inhibit cell viability and induce apoptosis in both AGS and HGC-27 gastric cancer cell line. Through high-throughput RNA sequencing, capsaicin exhibited inhibiting role in DNA repair, DNA replication and chromosome assemble pathway. qPCR assay and western blot validated that capsaicin could inhibit expression of the key enzymes (FEN1, LIG1 and PARP1) in DNA damage response and chemotherapy resistance. In vitro assay demonstrated that capsaicin could significantly induce cyto-toxicity and chemotherapy-induced DNA damage of 5-FU and Oxalipatin at low dose. Immunofluorescence analysis revealed that capsaicin inhibited both the expression and nuclear accumulation of PARP1 in AGS cells treated with 5-FU or oxaliplatin.</p> Conclusions <p>Capsaicin could inhibit DNA repair, thereby inhibited cell viability and improved the sensitivity of chemotherapy in gastric cancer cells.</p>