In continuation of our search for bioactive secondary metabolites from terrestrial Bacillus spp., a new microbial diketopiperazine, cis-cyclo-(His,Leu) (1) was isolated from the ethyl acetate extract of a strain B. subtilis B38, together with cis-cyclo-(Phe,Phe) (2), tryptophane (3), cis-cyclo-(Leu,Tyr) (4), cis-cyclo-(Trp,Tyr) (5) and macrolactin A (6). The chemical structures of the isolated compounds were identified by comparison of their 1D, 2D NMR and HRESIMS data with authentic spectra and literatures. To the best of our knowledge, this is the first time that cyclo-(His,Leu) has been isolated from natural products.