Pancreatic ductal adenocarcinoma (PC) is characterized by a highly fibrotic desmoplastic stroma. Subtypes of cancer-associated fibroblasts (CAFs) have been identified in chemotherapy-naïve PC (CTN-PC), but their precise functions are still unclear. Our knowledge regarding the properties of CAFs in the regressive stroma after neoadjuvant treatment (NAT) of PC (NAT-PC) is particularly limited. We aimed to examine the marker phenotypic properties of CAFs in the regressive stroma of PC. Surgical specimens from patients with CTN-PC (n=10) and NAT-PC (n=10) were included. Juxtatumoural, peripheral, lobular, septal, peripancreatic, and regressive stromal compartments were manually outlined using digital imaging analysis (DIA) for area quantification. The compartment-specific expression of CD271, cytoglobin, DOG-1, miR-21, osteonectin, PDGF-Rβ, and tenascin C was evaluated by immunohistochemistry or in situ hybridization, using manual scoring and automated DIA. The area fraction of the regressive stroma was significantly higher in NAT-PC than in CTN-PC (P=0.0002). CD271 (P<0.01), cytoglobin (P<0.05), DOG1 (P<0.05), miR-21 (P<0.05), and tenascin C (P<0.05) exhibited significant differences in their expression profiles between the juxtatumoural compared to the peripheral and regressive stroma. PDGF-Rβ expression was significantly higher in juxtatumoural than in peripheral CAFs (P<0.05). Our data provide further support of the concept of stromal heterogeneity and phenotypic different CAF subtypes in PC. CAFs in the regressive stroma of NAT-PC show a marker phenotype similar to some (namely, peripheral) and different from other (namely, juxtatumoural) previously defined CAF subtypes. It may be hypothesized that phenotypic CAF subtypes, at least in part, also may share functional properties. Studies examining the precise functional characteristics of CAF subtypes in PC are needed.