2′-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA–DNA and DNA–RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA–RNA duplexes similar to the one of the parent DNA–RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed ΔG values (37 °C) that are in line with the measured T_m values for both the DNA–DNA and DNA–RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA–DNA and DNA–RNA duplexes involving Un are similar to those of the dT–rA and dU–rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA–DNA and RNA–DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2′-exo sugar pucker conformation close to an A-helix type without perturbing the C2′-endo sugar pucker conformations of their 2′-deoxynucleoside neighbours. These results confirm the potential of 2′-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.