Background and Aims The signaling pathways associated with phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) and SMAD family member 4 (SMAD4) are closely linked to pancreatic adenocarcinoma (PAAD). However, the relationships of the two genes with the occurrence or prognosis of PAAD are not fully understood. This study was conducted to investigate the expressions PIK3C3 and SMAD in PAAD and their influences on prognosis of patients.Methods The mRNA expressions of PIK3C3 and SMAD4 in PAAD tissue and their corresponding effects on prognosis of PAAD patients were analyzed using UALCAN website. The mRNA expression profiles of PIK3C3 and SMAD4 together with the indicated clinicopathologic data were downloaded from TCGA. The PAAD patients were divided into two clusters based on the combined expression of the PIK3C3 and SMAD4 genes by consensus clustering analysis, the differences in PIK3C3 mRNA or SMAD4 mRNA expression pattern, clinicopathologic variables and overall survival between two clusters were analyzed by Kolmogorov-Smirnov test and Kaplan-Meier method. The protein levels of PIK3C3 and SMAD4 in tissue microarrays containing PAAD and adjacent normal tissue samples were determined by immunohistochemical staining, and the differences of the two proteins in tumor and adjacent normal tissue were evaluated by independent sample t test as well as the area under ROC curve (AUC); the effects of individual PIK3C3 and SMAD4 protein or their combination on the survival of PADD patients were assessed by Kaplan-Meier analysis.Results The results of analysis showed that mRNA expression of PIK3C3 or SMAD4 alone did not change significantly in PAAD and did not significantly affect the prognosis of PAAD patients as well (all P>0.05). There was a high correlation in co-expression between PIK3C3 mRNA and SMAD4 mRNA in PAAD. Between the two clusters of PAAD patients, there were significant differences in mRNA levels of the two genes and age (P<0.05), and the cluster with higher mRNA level of both PIK3C3 and SMAD4 had a better survival probability (P=0.006). The protein levels of both PIK3C3 and SMAD4 were significantly higher in PAAD tissue than those in adjacent normal tissue (both P<0.001), and their AUC were 0.7417 and 0.7991, respectively. PAAD patients with positive expression of PIK3C3 or SMAD4 protein have better survival than those with negative expression of both proteins (P=0.0359).Conclusion This study suggests that PIK3C3 and SMAD4 protein may be the potential biomarkers for diagnosis of PAAD. Combined analysis of PIK3C3 and SMAD4 can be used for predicting the prognosis of PAAD patients.