Agricultural transformation represents one of the greatest threats to biodiversity, causing degradation and loss of habitat, leading to changes in the richness and composition of communities. These changes in richness and composition may, in turn, lead to altered species co-occurrence, but our knowledge of this remains limited. We used a novel co-occurrence network approach to examine the impact of agricultural transformation on reptile community structure within two large (> 172 000 km2; 224 sites) agricultural regions in southeastern Australia. We contrasted assemblages from sites surrounded by intact and modified landscapes and tested four key hypotheses that agricultural transformation leads to (H1) declines in species richness, (H2) altered assemblages, (H3) declines in overall co-occurrence, and (H4) complex restructuring of pairwise associations. We found that modified landscapes differed in composition but not richness compared with intact sites. Modified landscapes were also characterized by differences in co-occurrence network structure; with species sharing fewer sites with each other (reduced co-occurrence connectance), fewer highly-connected species (truncation of the frequency distribution of co-occurrence degree) and increased modularity of co-occurrence networks. Critically, overall loss of co-occurrence was underpinned by complex changes to the number and distribution of pair-wise co-occurrence links, with 41–44% of species also gaining associations with other species. Change in co-occurrence was not correlated with changes in occupancy, nor by functional trait membership, allowing a novel classification of species susceptibility to agricultural transformation. Our study reveals the value of using co-occurrence analysis to uncover impacts of agricultural transformation that may be masked in conventional studies of species richness and community composition.