<p>Small cell lung cancer (SCLC) is a type of high-grade neuroendocrine malignancy with low gene mutation. Chemotherapy is the major treatment strategy, but long-term clinical application often leads to drug resistance. Etoposide is a first-line drug approved by the US Food and Drug Administration for SCLC treatment, but etoposide-resistance is a problem. In this study, a SCLC cell line with etoposide-acquired resistance, H1048-ER, was constructed through a concentration gradient increasing method, and its resistance to etoposide was investigated in vitro and in a zebrafish model. Through transcriptome sequencing, real-time reverse transcription-quantitative polymerase chain reaction, and bioinformatic analyses of H1048-ER <i>vs.</i> H1048 cells, 51 differentially expressed genes were found to be significantly enriched in “collagen degradation” and “MET/FAK signaling activation in ECM”. Among them, six genes (<i>COL11A1</i>, <i>COL26A1</i>, <i>COL4A3</i>, <i>COL4A4</i>, <i>LAMA4</i>, and <i>LAMC1</i>) had strong correlations with the prognosis of lung cancer. They may be key factors in the acquired etoposide resistance of H1048-ER cells. H1048-ER cells showed cross-resistance to cisplatin but were sensitive to doxorubicin and temozolomide. Our study provides novel insights into etoposide resistance in SCLC and affords the potential treatment options after etoposide resistance.</p>