Urinary tract infections (UTIs) are caused by both Gram-negative bacteria such as Escherichia coli and Gram-positive organisms, including staphylococci and streptococci. Global host responses triggered in the bladder following infection due to these organisms have been largely uncharacterized on a genome-wide scale. Here, we used two model causal organisms, uropathogenic E. coli (UPEC) and uropathogenic Streptococcus agalactiae (UPSA) to map the global bladder transcriptome of acute UTI in mice using Affymetrix Gene-1.0ST microarrays, and compared these responses to host-pathogen interactions that occurred between these model organisms and human bladder cells in vitro. Significant-gene lists were compared at 2h and 24h following infection in side-by-side infection comparisons with mock-infected female C57BL/6 mice. No genes exhibited significantly altered expression at 2h in UPSA-infected mice despite high bladder bacterial loads at this early time point. The absence of a marked early host response to UPSA juxtaposed with broad-based bladder responses activated by UPEC at 2h that encompassed over 1500 genes. In vivo bladder response data were interpreted against comparative in vitro binding assays using human bladder cells to determine if differences in bladder binding may explain the different transcriptional responses. However, in vitro assays showed that UPSA adhered to uroepithelial cells equally as rapidly (within 30 min) and efficiently as compared to UPEC. Furthermore, both organisms were able to invade these cells in vitro, and survive under intracellular conditions for at least 24h. In escalating challenge dose assays in mice, UPSA also demonstrated equivalent binding to the bladder uroepithelium in vivo compared to UPEC. Bioinformatic analyses showed that both organisms significantly activated biological pathways for regulation of leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. Overall, these data emphasize the pathogen-specific nature of bladder immune activation during acute UTI in vivo, despite similar in vitro bladder cell binding efficiencies to human cells.