Degradation or the removal of aflatoxin B1 from agriculture commodities is very important because of its acute toxicity and economic loss due to rejection of about 25% contaminated agri produce. The present study aimed at using Pseudomonas putida for the aflatoxin B1 (AFB1) degradation and to understand the mechanism involved. AFB1 degradation was studied with P. putida culture, culture supernatant, cell lysate, cell lysate in the presence of protease inhibitor, and heat-inactivated cell lysate. The remaining AFB1 was qualitatively and quantitatively measured by thin-layer chromatography and HPLC with a UV detector. P. putida culture and culture supernatant showed 80% reduction in AFB1 within 24 h of incubation. Cell lysate and the lysate in the presence of protease inhibitor showed the same reduction in 6 and 4 h respectively. The protease-inhibited lysate showed greater thermostability, broad pH range, and tolerance to some of the solvents and detergents in terms of aflatoxin B1 degrading activity. The heat-inactivated lysate showed only 20% reduction in 24 h of incubation indicating loss of activity on heating. As cell-free supernatant and cell lysate are capable of reducing AFB1 effectively, actively growing cells are not necessary for degradation. The active principle for degradation might be proteinaceous; therefore, heat-inactivated lysate is ineffective for reducing the AFB1. These results showed that degradation of aflatoxin B1 by P. putida might be an enzymatic process and could be used in a broad range of conditions. © 2019, Sociedade Brasileira de Microbiologia.