The apurinic/apyrimidinic (APE)-like endonuclease (EN) of the long interspersed element 1 (LINE-1, L1) initiates retrotransposition by nicking double-strand DNA at a consensus 5′-TTTT↓AA sequence, creating a 3′-OH group that primes L1 cDNA polymerization through a mechanism termed “target primed reverse transcription” (TPRT). The consensus motif reflects the site of completed retrotransposition events, which depend on A-T base pairing between the cleaved genomic DNA and the L1 RNA poly(A) tail. Therefore, the consensus may not represent intrinsic properties of L1 EN, and it remains unknown whether L1 EN also cuts other sequences. To investigate both canonical and non-canonical cutting activities of L1 EN, we purified monodisperse WT, E43S-D145N double mutant, and D145N-H230A double mutant enzymes to investigate potential non-canonical functions of WT. We have verified conventional nicking activity on a circular plasmid, and demonstrated that the E43S, D145N double mutant ablates cutting activity as expected. Here, we demonstrate a robust, non-canonical cutting activity of L1 EN. Secondary structure analysis and cutting activity on a variety of oligonucleotide fragments reveals that L1 EN favors cutting at positions adjacent to mismatched duplex DNA. This activity may be responsible for aspects of resolving TPRT intermediates, and we plan to expand this study to explore the role of L1 EN in second strand cutting in TPRT.