The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor‐restraining and tumor‐promoting fibroblast subpopulations. Re‐education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA‐21 (miR‐21) in K‐Ras‐driven p53‐deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR‐21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor‐restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR‐21 loss. Stromal miR‐21 activity was required for induction of tumor‐restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR‐21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor‐suppressive function of miR‐21 in in vivo PDAC models. These results have important clinical implications for anti‐miR‐21‐based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell‐intrinsic role of miR‐21 in cancer‐associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR‐21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC. What's new? The opposing tumor‐restraining and tumor‐promoting role of distinct subpopulations of cancer‐associated fibroblasts (CAFs) has emerged as a new paradigm in the biology of pancreatic cancer ductal adenocarcinoma (PDAC). In addition, microRNA‐21 (miR‐21), expressed in CAFs, is frequently upregulated in PDAC. This preclinical study in genetically engineered PDAC mice shows that global loss of profibrotic TGF‐β‐inducible miR‐21 is required for the activation of tumor‐restraining fibroblasts. The data reveal a profibrotic yet overall tumor suppressive function for miR‐21 in vivo. The findings are relevant for anti‐miR‐21‐based therapeutic strategies currently under investigation for various cancer types, including PDAC.