Eleven new pyrimidine nucleosides (<b>1</b>-<b>11</b>) and 12 known analogues (<b>12</b>-<b>23</b>) were isolated from the marine-derived <i>Streptomyces</i> sp. SSA28. All of the new structures were elucidated by extensive NMR spectroscopic analysis and HRESIMS data. The absolute configurations of compound <b>1</b> were determined by X-ray diffraction. The configurations of <b>2</b>-<b>16</b> were investigated by ECD calculations. Compounds <b>11</b>-<b>16</b> showed cytotoxicity against HCT-116 human colon cancer cell lines with IC<sub>50</sub> values from 0.39 ± 0.03 to 6.63 ± 0.47 μM.