Agave fibers were used to elaborate transparent and flexible cellulose hydrogel films used as a scaffold for tissue regeneration and tested by in vitro assays with NIH 3T3 fibroblast cells. Using a dimethylacetamide/lithium chloride (DMAc/LiCl) system made it 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 %. The resultant hydrogel films showed water contents in the range of 239-323% and enough film strength from 50 to 66 N/mm(2), when the LiCl was changed from 4 to 12 wt % concentrations, respectively. The prepared agave cellulose films showed better cytocompatibility than the polystyrene dish used as control. Atomic force microscopy 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.