AbstractPhosphoinositide-3-Kinase Regulatory Subunit 1 (PIK3R1) is believed to function as a tumor suppressor, while Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) as a tumor driver. However, there is no systematic pan-cancer analysis of them. The pan-cancer study comprehensively investigated the gene expression, genetic alteration, DNA methylation, and prognostic significance of PIK3R1 and PIK3R2 in 33 different tumors based on the TIMER, GEPIA, UALCAN, HPA, cBioPortal, and Kaplan–Meier Plotter database. The results indicated that PIK3R1 is lowly expressed in most tumors while PIK3R2 is highly expressed in most tumors, and abnormal gene expression may be related to promoter methylation. Moreover, not only mutations, downregulation of PIK3R1 and upregulation of PIK3R2 were found to be detrimental to the survival of most cancer patients as well. Furthermore, the expression of both PIK3R1 and PIK3R2 was associated with the level of immune infiltration in multiple tumors, such as breast invasive carcinoma. Our study conducted a comparatively comprehensive analysis of the role of PIK3R1 and PIK3R2 in a variety of cancers, contributing to further study of their potential mechanisms in cancer occurrence and progression. Our findings suggested that PIK3R1 and PIK3R2 could serve as prognostic markers for several cancers.