The biosilicification of diatoms allows for the customization of the synthesis of functionalized diatom frustules. The S active sites (–SH) on diatom frustules were created by adding the organic silicon sources tetramethoxysilane (TMOS) and (3-mercaptopropyl)trimethoxysilane (MPTMS). The mechanisms of adsorption-reduction and the indirect effects of S active sites on electrochemical performance were declared. The DBS@C-Ag-3 anode material sourced from the cultivation condition with a silicon source of TMOS:MPTMS = 3:1 shows the best comprehensive performance and delivers a discharge capacity of ∼660 mAh·g−1 after 1000 cycles at 1 A·g−1. The electrochemical performance of DBS@C-Ag anode materials is also found to be dominated by structure at high temperatures and conductivity at low temperatures. Such a diatom frustule structure with sulfhydryl functionalization is promising for anode materials, and it suggests a biological strategy for creating other electrode materials by modifying them with metals to improve electrochemical performances. Display omitted •-SH active sites on diatom frustules were created by adding organic silicon sources•Thiol-functionalized diatoms plus Ag modification are used as anode material sources•DBS@C-Ag-3 anodes delivered ∼660 mAh·g−1 after 1000 cycles at 1 A·g−1•Dominant role of structure and conductivity at different temperatures was revealed Electrochemistry; Materials science; Materials chemistry