Aims: Efficient manipulation of multiple regions within a genome can be improved by counter-selection approaches. In this work, we sought to develop a method to manipulate Azotobacter vinelandii using a counter-selection approach based on the presence of the pyrF gene. Methods and Results: A background uracil auxotroph of A. vinelandii was first constructed by deleting the pyrF gene coding orotidine-5'-phosphate decarboxylase. The pyrF gene and promoter were also incorporated together with an antibiotic marker to create a selection and counter-selection cassette to shuttle into various plasmids. The constructed cassette could then be removed using a plasmid lacking the pyrF gene via counter-selection resulting from the production of 5-fluorouracil. The process could be repeated multiple times using the same procedure for selection and counter-selection. Following completion, the pyrF gene may be reintroduced to the genome in its original location, leaving a completed strain devoid of any antibiotic markers. Conclusions: Utilization of the pyrF gene for counter-selection is a powerful tool that can be used effectively to make multiple gene deletions in A. vinelandii. Significance and Impact of the Study: This study demonstrates the successful application of a counter-selection approach to yield markerless genetic modifications to A. vinelandii, which should be of interest for a range of applications in this important model bacterium.