the present investigation was conducted to characterize enzymatic and molecular aspects of a beta 1 and 4 endoglucanase gene in Bacillus subtilis strain B5d.The gene was amplified with specific primers and its sequence was deposited in the NCBI (accession number ofKF670724).  Sequence and phylogenetic analyses were performed using the Vector NTI and Mega 4 software. The PCR product comprised of 794 bp, encoding a peptid of 264 amino acids in length. Amino acid sequence analysis showed that the gene encoding beta 1 and 4 endoglucanase in B. subtilis B5d had multiple domains including cellulose binding domain belongs to family 3 and catalytic domain belong to glycoside hydrolase family 5.The enzyme activity was assessed in temperature range of 30-70 °C and pH 4.6. The results showed that the maximum catalytic activity of the enzyme occurred at 55 °C and was estimated at 461.83 U/L. The stability of the enzyme was measured at different pH and temperatures values and the results showed that the enzyme was stable at pH 5-10 and temperature values up 60 °C.