Agave fibers were used to elaborate a transparent and flexible cellulose hydrogel films used as scaffold for tissue regeneration and tested by in vitro assays with NIH 3T3 fibroblast cells. Using dimethylacetamide/lithium chloride (DMAc/LiCl) system was possible to obtain cellulose solutions and hydrogel films were prepared by phase inverse method without cross-linker. The concentration of LiCl in the DMAc solution was varied from 4 to 12 wt% in the phase inversion process and then the cytotoxicity was tested for 14 days on the cultivation. The resultant hydrogel films showed better cytocompatibility than the PS dish used as control. The cell growing images showed that the hydrogel films with lower LiCl apparently contained ordered and aggregated fiber orientation. This comparison suggested that the segmental microstructure in the hydrogel films influenced fibroblast cells spreading. In addition, the agave hydrogel films displayed good stability without biodegradiation through the cell cultivation.