Oogenesis is a complex developmental process responsible for the production of eggs from oogonia in fish and other animals. However, transcriptional regulation underlying oogenesis is not fully understood. In the present study, we demonstrated in the teleost fish Nile tilapia that the Sox transcription factor family member Sox3 was involved in regulating oocyte growth during oogenesis. Fluorescence in situ hybridization showed that Sox3 expression was enriched in growing oocytes of ovary but could not be detected in testes. CRISPR/Cas9-mediated homozygous mutation in the Sox3 gene disrupted oocyte growth. Further analysis revealed that Sox3 mutation caused a decrease in the contents of neutral lipids in oocytes and estradiol-17 beta (E2) production. RNA-seq-based transcriptome profiling and RT-qPCR analysis in ovaries demonstrated that the expression levels of genes involved in E2 production, lipid accumulation, and yolk formation were significantly downregulated following Sox3 mutation. Altogether, our findings indicate that Sox3 is required for oocyte growth in Nile tilapia and provides insights into transcriptional regulation underlying oogenesis in teleost fish.