ABSTRACT Transposon insertion sequencing (TIS; also known as TnSeq) is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS) protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt) and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences. IMPORTANCE Transposon insertion sequencing (TIS) is often used to assess the relative frequency with which genetic loci can be disrupted, which is taken as an indicator of their importance for bacterial fitness. Here, we report that biological factors other than the relative levels of fitness of insertion mutants can influence TIS output. We found that the presence of the DNA binding protein H-NS, which preferentially recognizes AT-rich sequences, is linked to significant underrepresentation of mutations within AT-rich loci in transposon insertion libraries. Furthermore, there is a marked correspondence between loci bound by H-NS and loci with an increased frequency of disruption in a Δ hns insertion library relative to a wt library. Our data suggest that factors other than genetic fitness (e.g., DNA binding proteins such as H-NS) can systematically modulate the frequency of transposon insertions in TIS studies and add a note of caution for interpretation of TIS data.