Guide RNAs (gRNAs) are short mitochondrially encoded RNAs that contain the information for editing of messenger RNAs in Trypanosoma brucei. Although a great deal of work has focused on the utilization of gRNAs in editing, little is known about the turnover of gRNAs. In this report, we utilized in organello pulse chase and in vitro RNA decay experiments to directly examine gRNA turnover. We found that gRNAs are degraded by a biphasic mechanism. In the first step of decay, 3' gRNA sequences encompassing primarily the post-transcriptionally added oligo(U) tail are rapidly removed. This is followed by a second step, which entails a comparatively slower degradation of the encoded gRNA body. Decay of the 3' end of the gRNA is sequence specific, as it does not occur on oligoadenylated gRNAs. In contrast, the nucleotide composition of the 3' extension does not affect the rate of degradation during the second, slower, decay step. Finally, competition assays suggest that complete gRNA decay is mediated by two distinct enzymes, one of which simultaneously recognizes elements of the oligo(U) tail and the encoded portion of the gRNA. Overall, these results provide the first evidence for a gRNA-specific decay pathway.