The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1’s cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development. Display omitted •RNA-BisSeq revealed a dynamic RNA m5C landscape during zebrafish embryogenesis•Ybx1 preferentially recognizes m5C-modified mRNAs•Ybx1 deficiency leads to early gastrulation defects in zebrafish embryos•Ybx1 and Pabpc1a coordinately regulate m5C-modified maternal mRNA stability RNA modifications exert important effects in many critical physiological processes. Using RNA-BisSeq, Yang et al. provide a comprehensive view of the RNA m5C landscape in zebrafish early embryos and show that m5C-modified maternal mRNAs are stabilized by Ybx1 and Pabpc1a during zebrafish MZT.