It has previously been reported that high mobility group box chromosomal protein 1 (HMGB1) is overexpressed in the majority of gastric adenocarcinoma cell types, and that HMGB1 can be released into the extracellular matrix from stressed or necrotic cancer cells. HMGB1 is considered to promote cell proliferation and invasion in gastric adenocarcinoma cells. Furthermore, in a number of cancer cell types, HMGB1 has been reported to promote autophagy and inhibit anticancer drug-induced apoptosis, which has been identified as an important mechanism in the development of multidrug resistance (MDR). However, there have been no studies on the effects of HMGB1 on expression of the MDR-related transporter proteins in gastric adenocarcinoma. In the present study, extracellular HMGB1 increased the expression levels of P-glycoprotein (P-gp) at the pre-transcriptional and post-transcriptional levels in the human gastric adenocarcinoma cell lines, SGC7901, MKN28 and AGS, as detected by quantitative polymerase chain reaction and western blot assays. MTT and apoptosis assays were also performed and it was demonstrated that extracellular HMGB1 subsequently enhanced resistance to the P-gp-related drugs, adriamycin and vincristine. In brief, this study demonstrated that extracellular HMGB1 may promote drug resistance to adriamycin and vincristine by upregulating P-gp in human gastric adenocarcinoma cells.