Considering the great influence of tumour microenvironment on the development and metastasis of cancer progression, three dimensional (3D) culture models have become essential for improving cancer biology investigations as well as the development of novel anticancer drug screening approaches. The aim of this study was to develop a novel in vitro 3D model of pancreas cancer based on a human pancreatic cancer cell line BxPC-3 and a microstructured hydrogel made of chitosan (mCS) or a polyelectrolyte complex (mPEC) between CS and poly(γ-glutamic acid) (γ-PGA). The investigated hydrogels well supported the adhesion and proliferation of the cultured cells that were able to colonize the interconnected porous network of the 3D porous microstructures. As shown by confocal laser scanning microscopy (CLSM) analysis, BxPC-3 cells cultured in microstructured hydrogels maintained the cancer features typical of the cell line, such as loss of cell polarity and duct-like structures differentiation, and formed multicellular spheroids that were not observed in two-dimensional (2D) monolayer culture. Fascin protein, investigated as a marker of tumor invasiveness by CLSM microscopy and fluorescent western blotting technique, was found overexpressed in 3D tumor engineered constructs, unlike what is observed in 2D culture. In addition, cells cultured in combination with 3D hydrogels overexpressed a set of cytokines associated with pancreatic cancer invasiveness, as assessed by means of luminex technology. The obtained results suggested that the developed microstructured hydrogel/cell constructs represent a promising 3D tumor engineered model with great potential for the development of in vitro investigations providing innovative insights into pancreas carcinogenesis processes.