Animal experiments have been widely conducted in the life sciences for more than a century, and have long been a subject of ethical and societal controversy due to the deliberate infliction of harm upon sentient animals. However, the harmful use of animals may also negatively impact the mental health of researchers themselves. We sought to evaluate the anxiety level of researchers engaged in animal use to analyse the mental stress from animal testing. The State Anxiety Scale of the State-Trait Anxiety Inventory (STAI) was used to evaluate how researchers feel when they conduct animal, as opposed to non-animal, based experiments (95 non-animal and 98 animal testing researchers). The Trait Anxiety Scale of STAI was employed to measure proneness to anxiety, namely the base trait of the researchers. Additionally, the information on sex, age, education, income, and total working periods was collected. While the Trait Anxiety scores were comparable (41.5 ± 10.9 versus 42.9 ± 10.1, p = 0.3682, t-test), the State Anxiety scores were statistically significantly higher for animal users than non-animal users (45.1 ± 10.7 versus 41.3 ± 9.4, p = 0.011). This trend was consistent for both male and female. Notably, younger animal testers (≤ 30 years of age) with less work experience (≤ 2 years) and lower income level (≤ 27,000 USD) exhibited higher anxiety scores, whereas these factors did not affect the anxiety level of non-animal users. The present study demonstrated that participation in animal experiments can negatively impact the mental health of researchers.