This article overviews recent findings on molecular mechanisms of gene expression regulation published in the “New Sights into Bioinformatics of Gene Regulations and Structure” (https://www.mdpi.com/journal/ijms/special_issues/MVA479KFR7, accessed on 30 June 2025) Special Issue. Key approaches are based on bioinformatics methods, high-throughput sequencing, and post-genome technologies. We have initiated this Special Issue based on the discussions on bioinformatics of gene expression regulation at the transcription level presented at the “Bioinformatics of Genome Regulation and Structure/Systems Biology” (BGRS/SB) conference series in Novosibirsk, Russia. Begun in 1998, BGRS/SB is the longest-running biannual conference series gathering scientists, bioinformaticians, medical doctors, and geneticists in Novosibirsk, Russia (https://bgrssb.icgbio.ru/2024/, accessed on 30 June 2025). IJMS has already published several successful Special Issues on the bioinformatics of gene expression, including the following: “Molecular Mechanisms of Gene Expression: ‘Bioinformatics of Gene Regulations and Structure'” (2020), “Bioinformatics of Gene Regulations and Structure—2022” (https://www.mdpi.com/journal/ijms/special_issues/Bioinformatics_Gene, accessed on 30 June 2025), and “25 Anniversary of Bioinformatics of Genome Regulation and Structure Conference Series” (https://www.mdpi.com/journal/ijms/special_issues/0LGA6103S5, accessed on 30 June 2025), as well as earlier Special Issues [1,2]. The BGRS conference continues initiating series of science discussions on perspective research fields such as interdisciplinary studies in computational genomics of model organisms—plants, animals, bacteria, complex network interactions, and the systems biology approaches for gene transcription analysis, including machine learning and AI (artificial intelligence) tools