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a-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase ? (hPol ?) opposite a TNA-modified template strand without and in combination with O⁴-alkyl thymine lesions. Across TNA-T (tT), hPol ? inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol ? inserted dAMP opposite O⁴-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol ? complexes with template tT and O⁴-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' ? 2' versus 5' ? 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 Å on average from the a-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol ?. Conversely, the crystal structure of a ternary hPol ?·DNA·tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP a-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.