As one of the important traits in pig production, meat quality has important research significance and value. Intramuscular fat (IMF) content is one of the most important factors affecting pork quality. Many experimental studies have shown that IMF content is closely related to the flavor, tenderness, and juiciness of pork. Therefore, it is of great significance to study the mechanism of porcine IMF deposition. Previous research indicated that miR-149-5p promoted the proliferation of porcine intramuscular (IM) preadipocytes and decreased their ability to differentiate, albeit the exact mechanism of action is unknown. In vitro, foreign pigs showed increased miR-149-5p expression and reduced fat deposition when compared to Queshan Black pigs. This study conducted metabolomics and transcriptomics analyses of porcine IM preadipocytes overexpressing miR-149-5p to verify their effects on lipid formation. According to metabolomics analysis, the overexpression of miR-149-5p has significantly altered the lipid, organic acid, and organic oxygen metabolites of porcine IM preadipocytes. Specially speaking, it has changed 115 metabolites, including 105 up-regulated and 10 down-regulated ones, as well as the composition of lipid, organic acid, and organic oxygen metabolism-related metabolites. RNA-seq analysis showed that overexpression of miR-149-5p significantly altered 857 genes, of which 442 were up-regulated, and 415 were down-regulated, with enrichment to MAPK, IL-17, PI3K-Akt, and ErbB signaling pathways. We found that overexpression of miR-149-5p inhibited adipogenic differentiation by changing cAMP signaling pathway in porcine IM preadipocytes. In addition, the overexpression of miR-149-5p may affect the transport of Cu2+by targetingATP7Aand inhibiting adipogenic differentiation. These findings elucidate the regulatory function of miR-149-5p in porcine IM preadipocytes, which may be a key target for controlling pork quality.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09382-6.