Background: Immunotherapy has improved outcomes for many patients (pts) with advanced cancers found to be microsatellite instability high (MSI-H). However, not all MSI-H pts exhibit the same efficacy from immunotherapy. Previous studies have suggested that concomitant antibiotic use while receiving immunotherapy may result in poorer outcomes. We aimed to evaluate what factors impacted immune checkpoint blockade (ICB) response in pts with MSI-H metastatic colorectal cancer (mCRC) treated at a large academic referral center. Methods: We completed a single-site, retrospective review of 45 pts with MSI-H mCRC that received ICB. Data was collected including patient demographics, tumor and mutational data, ICB agent, length of treatment, and antibiotic use. Both single and multi-course antibiotic use was included from 2 weeks prior until 6 weeks following ICB treatment. Fisher’s exact test was used to evaluate the association of response and covariables. Log rank test was used to compare progression free survival (PFS) between subgroups. Results: A majority of pts were male (n = 25, 56%) and Caucasian (n = 30, 66%) with right sided tumors (n = 26, 62%). Combination Nivolumab and Ipilimumab was used in 7 (16%) with single agent Nivolumab in 10 (22%) and single agent pembrolizumab in 28 (62%). Of the pts in this cohort, 28 (62%) received antibiotics with 11 (39%) receiving a single round of antibiotics and 17 (61%) receiving multiple courses. Those that did not receive antibiotics had a greater response (partial or complete) at first scan while on ICB therapy (75% vs 32%, p = 0.01). No significant difference was noted between class of antibiotic, age, sex, or tumor sidedness. The median 12 month PFS (12-PFS) was also higher in the group that did not require antibiotics but was not statistically significant (82% vs 72%, p = 0.298). Conclusions: This study shows that MSI-H mCRC pts that received antibiotics while on ICB had impaired response rate and a trend towards lower PFS. This suggests alteration of the gut microbiome may impact ICB response warranting a larger, prospective study.