Objective: To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance. Methods: Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was estab-lished by the method of repeated clinical serous peak concentration plus gradually increasing concentration of gemcitabine from its parental cell human lung adenocaroinoma cell line A549 which was sensitive to gemcitabine. During the course of inducement, we had monitored their morphology, checked their resistance indexes and resistant pedigree by MTT method, gathered their growth curves and calculated their doubling time, examined their DNA contents and cell cycles by FCM; at the same time, we had measured their expressions of P53, EGFR, Cerb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, and CD44v6 proteins via immunocytochemistry staining, RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR. Results: The resistance index of A549/Gem' cells (the deputy of cells in the process of inducement) to gemcitabine was 163.228, and the cell line also exhibited cross-resistance to vinorelbine, taxotere, fluorouraci, etoposide and cisplatin, but kept sensitivity to paclitaxol and oxaliplatin. The doubling time of A549/Gem' was shorter and figures in G0-G1 phases were increased than A549 cells. Compared with A549 cells, A549/Gem' cells achieved EGFR and c-myc proteins expressions, nm23 protein expression enhanced, P53, Cerb-B-2 and Bcl-2 proteins expressions reduced, PTEN ,PCNA and MDR-1 proteins expressions vanished, but those of MMP-9, VEGF, CD44v6 and TIMP-1 proteins changed trivially. Meanwhile, expressions of RRM1 and ERCC1 mRNA were augmented markedly. The resistance index of A549/Gem cells to gemcitabine was 129.783, and the cell line also held cross-resistance to vinorelbine, taxotere, etoposide, cisplatin and sensitivity to paclitaxol. But the resistance to fiuorouracil and sensitivity to oxaliplatin vanished. And the expression of RRM1 and ERCC1 mRNA decreased visibly. The doubling time of A549/Gem cells was longer and figures in G0-G1 phases were decreased than A549/Gem' cells. In A549/Gem cells, expressions of P53, EGFR, PCNA and MDR-1 proteins was same to those of A549/Gem' cells. A549/Gem cells achieved TIMP-1 and PTEN proteins expressions, Cerb-B-2, MMP-9, c-myc and Bcl-2 proteins expressions enhanced, nm23 protein expressions vanished, but the expressions of VEGF and CD44v6 proteins changed trivially. Furthermore, Compared with its parental cell A549, A549/Gem cell was mixed with giant cells of different sizes and was larger and more irregular. Conclusion: The human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells A549. And these changes possibly participated in the formation of multidrug resistance.