Rainbow trout (Oncorhynchus mykiss) is a globally important cold-water aquaculture species, yet the molecular events underlying the transition from embryo to free-living larva remain poorly resolved. In this study, eukaryotic reference-based transcriptome sequencing was used to compare genome-wide gene expression profiles between eggs (eyed-egg stage) and newly hatched larvae of O. mykiss. A total of 618 differentially expressed genes (DEGs) were identified, of which 297 were upregulated and 321 downregulated. Gene Ontology (GO) enrichment analysis showed that DEGs were significantly enriched in immune system process, response to stimulus, and biological regulation. Within the immune system process category, 4 DEGs were upregulated and 11 were downregulated in larvae, indicating a more complex remodelling of immune-related gene expression than simple activation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified only Glycolysis/Gluconeogenesis (map00010) as significantly enriched (padj < 0.05). Nitrogen metabolism (map00910) and tryptophan metabolism (map00380) showed enrichment trends but were not statistically significant after multiple-testing correction. Because these pathways contained only a small number of DEGs and displayed mixed regulation, no overall pathway-level up- or down-regulation was inferred. These results suggest a possible metabolic remodelling during the transition from yolk-dependent anabolism to catabolic utilization in larvae. Transcript assembly further identified 134,770 potential novel isoforms and 2902 intergenic transcripts classified as putative novel transcripts, enriching the genome annotation of O. mykiss. Trend clustering indicated coordinated regulation of immune and stress-response genes together with downregulation of amino acid and energy metabolism genes, revealing programmed temporal regulation during this developmental transition. These findings provide foundational data for understanding early development in rainbow trout and candidate molecular targets for health-oriented aquaculture management.