The evolutionary development of adventitious roots (ARs) in plants enhances their capacity to adapt to various stress conditions. A thorough analysis of the influencing factors in their morphological construction holds significant theoretical value and practical guidance for overcoming rooting obstacles in cuttings, as well as for cultivating superior varieties characterized by broad adaptability and stress resistance. In this study, we investigated the molecular mechanisms underlying the development of ARs in tomato (Solanum lycopersicum) by performing transcriptome sequencing (RNA-seq). We analyzed the transcription profiles of relevant genes in the “Y962” strain, which exhibits spontaneous AR formation, and the “W961” strain, which does not form ARs. Our findings indicate that the AR induction stage represents an active phase of development, during which we identified 1,676 overlapping genes across the three comparison groups, highlighting the most differentially expressed genes. Functional enrichment analysis showed that they were most closely related to response to auxin, and were also dependent on the crosstalk between other hormones and carbohydrates. Furthermore, through the measurement of endogenous auxin levels and the induction tests with exogenous auxin, it was established that the formation of ARs is closely linked to the accumulation and transport of auxin. Notably, the auxin efflux SlPIN3, which was enriched in the auxin response pathway, exhibited significantly high expression during the induction phase of ARs. The slpin3 mutant, generated using the CRISPR/Cas9 editing system, exhibited a significant reduction in the number of ARs, highlighting the close relationship between polar transport regulated by SlPIN3 and auxin-induced AR formation. In summary, this study not only enriches the developmental network of AR formation in tomatoes with a wealth of data but also elucidates the potential mechanisms for promoting AR development by targeting SlPIN3.