Exposure to multiple stress factors is believed to contribute to honey bee colony decline. However, little is known about how co-exposure to stress factors can alter the survival and behavior of free-living honey bees in colony conditions. We therefore studied the potential interaction between a neonicotinoid pesticide, thiamethoxam, and a highly prevalent honey bee pathogen, Deformed wing virus (DWV). For this purpose, tagged bees were exposed to DWV by feeding or injection, and/or to field-relevant doses of thiamethoxam, then left in colonies equipped with optical bee counters to monitor flight activity. DWV loads and the expression of immune genes were quantified. A reduction in vitellogenin expression level was observed in DWV-injected bees and was associated with precocious onset of foraging. Combined exposure to DWV and thiamethoxam did not result in higher DWV loads compared to bees only exposed to DWV, but induced precocious foraging, increased the risk of not returning to the hive after the first flight, and decreased survival when compared to single stress exposures. We therefore provided the first evidence for deleterious interactions between DWV and thiamethoxam in natural conditions.