Abstract5‐Azacytosine (5‐AC) is one of the best interesting noncanonical nucleobases due to its functionalization and structural imitation of natural bases. 5‐AC can be used as the scaffold of two important chemotherapeutic medicines, 5‐azacytidine and 2′‐deoxy‐5‐azacytidine. Furthermore, increased sensitivity to UV leads to the photochemical effects of 5‐AC also attracted attention. Yet, no study has been reported to explore the effect of glycosyl groups on the photophysical and photochemical properties of 5‐AC, which can help to reveal the photostability of related actual clinic drugs. In this study, the excited‐state dynamics of 5‐azacytidine and 2′‐deoxy‐5‐azacytidine are studied by femtosecond transient absorption and quantum‐chemical calculations while revisiting that of 5‐AC with a wider probe spectral range. It is shown that glycosyl substitution on the N1 position leads to ultrafast excited‐state relaxation within several picoseconds in both nucleosides, which is distinct compared with the 17 ps lifetime seen in 5‐AC. It is proposed that these changes are due to altering the energy level of the dark nπ* state. Moreover, our results suggest that it should be cautioned to simply replace sugar groups with methyl groups when doing a theoretical calculation study on nucleobases and their derivatives.