Strains of Escherichia coli B, especially BL21, have been widely used for overproducing recombinant proteins, ethanol, and other biomolecules. Almost all laboratory strains of E. coli are derivatives of non-pathogenic K-12 or B strains. While most genetic and metabolic studies have been performed with K-12 strains, little has been done on B strains. Recently, genome sequences of two E. coli strains of the B lineage, REL606 and BL21(DE3), have been determined, and results of multi-omics analyses were compared between B and K-12. As compared to K-12, B strains show a number of phenotypes such as faster growth in minimal media, lower acetate production, higher expression levels of recombinant proteins, and less degradation of such proteins during purification. In this review, we summarize the unique biological features of the B strains and overview their academic and industrial applications.