The aims of the study were the production improvement, the purification, the characterization and the activity investigation of chitosanase CSNV26 of Bacillus subtilis (V26). The gene csnV26 encoding for this protein was amplified and cloned in the pBAD vector then expressed in Escherichia coli (Top10). The SDS-PAGE and zymogram analysis of the recombinant protein showed that it has two active forms sized 27 and 31 kDa, corresponding to the protein with and without signal peptide. This protein has the particularity of being secreted by Top10-pBAD-csnV26 with a high yield of 6.2 g/l. The HPLC purification of CSNV26 from supernatant confirmed the presence of the two sizes. The investigation of the CSNV26 thermostability showed that the pure protein is highly stable keeping 68 % of its activity after 30-min treatment at 100 °C, contrarily to the protein present within the supernatant of E. coli and B. subtilis (V26). The molecular dynamics study of the predicted structure of protein in both forms showed that the presence of the peptide signal in the form of 31 kDa gave it a remarkable thermal stability. The antifungal activity of CSNV26 was evidenced on Rhizopus nigricans and Rhizopus oryzae. Indeed, it has provoked an alteration and embrittlement of their hyphae with onset of protoplast.