Honey bees are important agricultural pollinators that rely on a specific gut microbiota for the regulation of their immune system and defense against pathogens. Environmental stressors that affect the bee gut microbial community, such as antibiotics and glyphosate, can indirectly compromise bee health. Most of the experiments demonstrating these effects have been done under laboratory conditions with pure chemicals. Here, we investigated the oral and topical effects of various concentrations of glyphosate in a herbicide formulation on the honey bee gut microbiota and health under laboratory and field conditions. Under all of these conditions, the formulation, dissolved in sucrose syrup or water, affected the abundance of beneficial bacteria in the bee gut in a dose-dependent way. Mark-recapture experiments also demonstrated that bees exposed to the formulation were more likely to disappear from the colony, once reintroduced after exposure. Although no visible effects were observed for hives exposed to the formulation in field experiments, challenge trials with the pathogen Serratia marcescens, performed under laboratory conditions, revealed that bees from hives exposed to the formulation exhibited increased mortality compared with bees from control hives. In the field experiments, glyphosate was detected in honey collected from exposed hives, showing that worker bees transfer xenobiotics to the hive, thereby extending exposure and increasing the chances of exposure to recently emerged bees. These findings show that different routes of exposure to glyphosate-based herbicide can affect honey bees and their gut microbiota.IMPORTANCE The honey bee gut microbial community plays a vital role in immune response and defense against opportunistic pathogens. Environmental stressors, such as the herbicide glyphosate, may affect the gut microbiota, with negative consequences for bee health. Glyphosate is usually sprayed in the field mixed with adjuvants, which enhance herbicidal activity. These adjuvants may also enhance undesired effects in nontargeted organisms. This seems to be the case for glyphosate-based herbicide on honey bees. As we show in this study, oral exposure to either pure glyphosate or glyphosate in a commercial herbicide formulation perturbs the gut microbiota of honey bees, and topical exposure to the formulation also has a direct effect on honey bee health, increasing mortality in a dose-dependent way and leaving surviving bees with a perturbed microbiota. Understanding the effects of herbicide formulations on honey bees may help to protect these important agricultural pollinators.