Abstract <p>Colorectal cancer (CRC) is a prevalent malignancy of the intestinal epithelium. Reticulon 2 (RTN2) plays a crucial role in the progression of various types of cancer. The aim of this study was to determine the correlation between RTN2 expression and clinicopathological characteristics in CRC cells, and to investigate the influence of RTN2 expression on endoplasmic reticulum (ER) Ca²⁺ levels. We then examined RTN2 expression in CRC, and RTN2 expression was upregulated in both CRC tissues and cells. Upregulated RTN2 expression was strongly correlated with the depth of infiltration, lymph node state, tumor stage, and degree of differentiation. Down-regulation of the expression of RTN2 inhibited proliferation, migration, and invasion of CRC (HCT116 and SW480) cells. Mechanistically, the downregulation of RTN2 expression was found to promote the acetylation of p53 in CRC cells. The p53 transcriptional activity inhibitor pifithrin-alpha (PFTα) reversed the regulatory effect of RTN2 on p53 acetylation. In addition, knockdown of RTN2 upregulated the mRNA expression of p53 target genes p21, NOXA, and Bax, and downregulated the mRNA expression of Bcl-2 in HCT116 and SW480 cells. PFTα reversed the inhibitory effect of RTN2 downregulation on proliferation, migration, and invasion of CRC cells. For ER Ca²⁺ levels, knockdown of RTN2 elevated Ca²⁺ levels and the expression of ER Ca²⁺ efflux-related factor IP3R via the p53 pathway. In summary, RTN2 promotes cell survival and decreases ER Ca²⁺ levels in CRC by activating the p53 signaling pathway. Consequently, RTN2 holds promise as a potential therapeutic target in CRC management.</p>