Pain is one of the most common symptoms in cancer patients, but for a long time, the control of pain symptoms in clinical cancer treatment still cannot get enough attention. The mechanism of pain promoting tumor has not been fully understood. Substance P (SP), as a neuropeptide mediating pain, is highly expressed in serum of patients with cancer pain. In this study, the SP stimulated tumor cells were collected and analyzed by synchrotron radiation microscopic infrared spectroscopy imaging, in order to reveal the possible mechanism of pain promoting tumor. Results show that in the breast cancer cell lines MDA-MB-231, MCF-7, and lung cancer cell lines A549, SPCA, the absorption peak of amide I near 1 655 cm^-1 which reflect the secondary structure of the protein, showed blue shift. Suggesting that SP stimulate tumor cells may cause tumor cell protein secondary structure change. In MDA-MB-231 and A549, the absorption peaks of amide I and amide II which located in 1 500~1 600 and 1 600~1 700 cm^-1 also declined significantly, show that in the two cell lines, the protein expression of tumor cells is significantly lower. In A549 cell line, the symmetric stretch vibration peaks of phosphodiester group in 1 070~1 090 and 1 230~1 250 cm^-1 show blue shift, indicating that SP stimulated A549 cell lines also affected the nucleic acid structure of this cell line, suggesting that SP may cause the damage of tumor suppressor genes and the activation of oncogenes. Synchrotron radiation micro-infrared imaging analysis revealed that persistent pain symptoms affect the biological activity of tumor cells through the release of neurotransmitters, possibly by changing the structure of proteins and nucleic acids in tumor cells. The results of spectroscopy showed that the presence of pain and other adverse symptoms and the release of pain-related substances may affect the protein structure and expression in tumor cells and the change of nucleic acid in tumor cells, which might have adverse effects on the treatment of tumors. In clinical tumor treatment, attention should be paid not only to killing tumor cells but also to symptomatic control of symptoms.