Background and purpose: Physical inactivity and prolonged sitting have associations with adverse health. University office workers are at a high risk of psychological and pain issues. This study aimed to explore the differences in sitting time, levels of physical activity participation, stress, depression, anxiety, and pain among university office workers in China and Australia. Methods: Online surveys were distributed to university office workers over 55 years at two universities in China and Australia, respectively. Results: A total of 185 participants completed the online survey (119 in China and 66 in Australia). Significant differences were found in sitting time during workdays between the two countries (p < 0.05) with a longer sitting time in the Australian respondents (7.5 h/day) than those in China (4.6 h/day). Additionally, there were also significant differences in terms of levels of depression and pain symptoms within the two countries (p < 0.05). The Australian respondents reported high levels of depression and pain (M = 7.38, SD = 5.86 and M = 3.65, SD = 2.21, respectively) than those in China (M = 5.71, SD = 4.87 and M = 1.89, SD = 1.89, respectively). The gender, education level, and sitting time of participants were found to be associated with pain scores (p < 0.05). A significant association between marital status and pain scores was found among the Australian respondents (p < 0.05). Conclusions: Future studies with a larger population are needed to validate the results and to further explore the association between physical activity participation and psychological wellbeing among university office workers.