Aims: To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. Methods and Results: Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N.punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3h. Interestingly, Cdf31(-) and Cdf33(-) mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31(-) mutant had a more profound effect on cdf33 expression than Cdf33(-) did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA(-)- and ZitB(-)-deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E.coli and the cyanobacterial transporters. Conclusions: The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N.punctiforme. Significance and Impact of the Study: This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.