© 2020 Informa UK Limited, trading as Taylor & Francis Group.In the current study, use of cost effective nitrogen source in the production of protease was aimed by using Bacillus subtilis ZBP4 which is an indigenous strain capable of producing various enzymes and industrially important metabolites. Lentil flour (LF) was used for this purpose, and enzyme production from LF was compared with some other nitrogen sources including casein, yeast extract, peptone from meat, skim milk powder, whey powder, and soy bean flour. Enzyme production in 48 h with LF (863 ± 21.4 U/mL) was lower than only yeast extract (2344 ± 24 U/mL) and soy bean flour (1424 ± 67.6 U/mL). On the other hand, it was considerably higher than all of the other nitrogen sources. Effect of LF concentration (5–20 g/L) was determined by using only LF as nitrogen source and the highest enzyme production was obtained at 20 g/L concentration (1000 ± 29.2 U/mL). Effects of pH (5–9) and temperature (30–45 °C) were also determined and the optima were found as pH 8 and 30 °C. As a consequence, LF can be a candidate as a nitrogen source for protease production by Bacillussubtilis ZBP4.