The aim of this study was to identify functional genes associated with the oviposition of Mytilus galloprovincialis and to elucidate the underlying mechanisms of oviposition. Transcriptomic analysis of female Mytilus galloprovincialis gonads was conducted before and after spawning. Pairwise comparisons of sequencing libraries from tissue samples were also performed. Before and after spawning, there were 4060 differential genes, with 1488 up-regulated and 2572 down-regulated genes. The GO functional enrichment terms are grouped into three functional categories: biological process, cellular component, and molecular function. Some genes were annotated by GO terms such as growth, developmental process, reproduction, and reproductive process. KEGG enrichment analysis revealed signaling pathways associated with oviposition and reproduction, including the TCA cycle, GnRH signaling pathway, ovarian steroidogenesis, estrogen signaling pathway, oxytocin signaling pathway, and steroid biosynthesis. In conjunction with GO and KEGG enrichment results, we identified five potential genes associated with reproduction: gonadotropin-releasing hormone receptor (GnRHR), luteinizing hormone β (LHβ), matrix metalloproteinase 14 (MMP14), cytochrome P4503A enzyme (CYP3A), and cytochrome P45017A enzyme (CYP17A). The expression levels of LHβ and MMP14 genes increased, whereas the expression levels of GnRHR, CYP3A, and CYP17A genes decreased. These findings were validated using real-time quantitative PCR (RT-qPCR). The differential expression of the five genes mentioned above is crucial in regulating the synthesis of sex hormones and reproductive process during the spawning of Mytilus galloprovincialis, which is consistent with the results of transcriptome sequencing. In this study, we utilized transcriptome sequencing technology to analyze the differences in the gonad transcriptome of female Mytilus galloprovincialis before and after spawning. The aim was to identify the key pathways and candidate genes involved in spawning, which could provide valuable data for the breeding of Mytilus galloprovincialis.