Limited attention has been given to the role of cultural orientation towards different ethnic groups in multi-ethnic settings without a dominant host culture. We evaluated whether acculturation levels, reflecting cultural orientation towards other ethnic groups, were associated with obesity and related lifestyle behaviors in a cosmopolitan Asian population. We conducted the current study based on data from the Singapore Multi-Ethnic Cohort (N = 10,622) consisting of ethnic Chinese, Malays, and Indians aged 21 to 75 years. Multivariable linear and logistic regression analyses were used to examine associations between the acculturation level (z-score), obesity, and related lifestyle behaviors, including dietary habits and physical activity. A higher acculturation level was directly associated with a higher prevalence of obesity among Chinese, whereas an inverse association was found for ethnic Indians, and no significant association in Malays. In ethnic Malays, greater acculturation was significantly associated with higher dietary quality and less sedentary time. Furthermore, a high acculturation level was significantly associated with higher sugar-sweetened beverage consumption and more leisure-time PA in all ethnic groups. Our findings suggest that greater cultural orientation towards other ethnic groups was associated with convergence in obesity levels. More research is required to understand how acculturation affects obesity-related lifestyle factors in multi-ethnic settings.