Distress or alarm calls are vocalizations made when animals are in stressful situations or faced with a predator. Squirrels (Sciuridae) are known for being very vocal; however, most studies on alarm vocalizations are limited to ground squirrels. We investigated the acoustic behavior of the arboreal fox squirrel (Sciurus niger) under different conditions. Specifically, we tested the hypothesis that fox squirrels modify acoustic alarm behavior in response to different perceived threat levels and that this response is affected by sex and individual experience. Squirrels were trapped, and acoustic data were collected during periods in which the squirrels were alone, approached by humans, manipulated in traps, and handled by humans. Calls were categorized based on acoustic features, and we quantified the call rate (calls/s) across conditions. Threat level significantly affected vocal rate, with squirrels producing more calls overall when alone but shifting the proportion of emitted call types as threat level increased. Sex, capture history, and individual had no effect on call rate. These results suggest that fox squirrels use a graded alarm call response system to respond to threatening situations.