The emerging spread of antibiotic resistance genes (ARGs) and their subsequent acquisition by clinically relevant microorganisms is a serious threat to human health. Animal manure has been recognized as an important reservoir of ARGs, however, the dissemination of manure-derived ARGs and the impacts of manure application on the soil resistome remain obscure. Here, we conducted microcosm studies to assess the temporal succession of a broad-spectrum of ARGs in two contrasting soils following manure application from cattle which had not been treated with antibiotics. High-capacity quantitative PCR detected 52 unique ARGs across all the samples, with β-lactamase as the most dominant ARG type. Several genes of soil indigenous bacteria conferring resistance to β-lactam, which could not be detected in manure, were found to be highly enriched in manure-treated soils, and the level of enrichment was maintained over the entire course of 140 days. The enriched β-lactam resistance genes had significantly positive relationships with the relative abundance of the integrase intI1 gene, suggesting an increasing mobility potential in manure-treated soils. The changes in ARG patterns were accompanied by a significant effect of cattle manure on the total bacterial community compositions. Our study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs. Our findings are important for reliable prediction of ARG behaviours in soil environment and development of appropriate strategies to minimise their dissemination.