In recent years, SRSF3 has been shown to be a proto-oncogene involved in a variety of tissue-specific splicing events and is closely related to human diseases. The splicing factor SRSF3, an important member of the serine- and arginine-rich protein family, is abnormally highly expressed in a variety of tumors and plays an important role in tumor cell proliferation, migration, and invasion. In this paper, in order to deeply analyze the role of SRSF3 in rectal cancer, RNA sequencing technology was used to determine its role in gene expression regulation. By preparing relevant reagents and materials, carrying out experimental work such as cell recovery, cell culture and passaging according to the steps, emphasizing the extraction of total intracellular proteins, we analyzed the expression of the shear factor SRSF3 in the tumor tissues of rectal cancer. The clinical case parameters of 60 rectal cancer patients were selected for correlation analysis, and the correlation coefficient between SRSF3 and clinicopathological parameters was less than 0.05, that is, the expression of SRSF3 was positively correlated with the pathological grading of colorectal tumors, while it was not correlated with other pathological features. Moreover, the expression of SRSF3 in rectal cancer tumor tissues was significantly higher than that in normal tissues, which could promote rectal cancer development by regulating the splicing of a series of cancer-related genes.