Rat tickling is a technique used by humans with rats to mimic rough-and-tumble play, improve welfare, and reduce fear. Anecdotal information suggests that rat tickling is also beneficial for humans, yet this assertion has not been empirically validated. We hypothesized that rat tickling would be beneficial to multiple populations interacting with or viewing rats at pet stores, including employees, customers, and new rat owners. We sampled nine employees, 806 customers, and 35 rat owners in two pet stores across three replicates. Employees were assessed after caring for rats (using tickling or minimal handling methods) and after the sale of each rat with the Animal Empathy Scale and the Positive and Negative Affect Scale. Customers were asked via a survey to determine which cage of rats they would purchase and which cage of rats looked the happiest. New rat owners' reasons for purchase, satisfaction, and attachment were assessed with surveys, including the Lexington Attachment to Pets Scale. Data were analyzed using general linear models (employees and owners) and generalized linear mixed models (customers). Employee affect was unaltered by handling treatment in the first four days. At the point of sale, employee positive affect was higher when selling controls versus tickled rats, pets versus feeders, and rats that had been in the store for a shorter time. Customers were more likely to identify tickled high-calling and control rats as being happier. Customers were more likely to choose rats from cages with a higher proportion of colored rats for purchasing. Owners and customers frequently cited behavioral reasons as important for selection of rats. In conclusion, based on the measures used in our study, short-term tickling of pet store rats may have minimal effects on humans. However, future research is warranted to investigate effects of more frequent or longer-term tickling.