The lack of a standardized reliable and valid instrument makes it difficult to measure attitudes toward lesbian women and gay men consistently and thus poses a challenge to compare and contrast intervention measures. This study aimed to validate Herek’s ATLG scale among undergraduates in mainland China and identify factors associated with negative attitudes toward LG. A total of 6,036 eligible undergraduates conveniently drawn from 30 provinces across mainland China were randomly split in half. Item analysis was first used to select (...) unrelated or redundant items for deletion. Exploratory factor analysis were then conducted on the first half of the sample, followed by confirmatory factor analysis and reliability analysis in the second half. Logistic regression analyses were finally carried out to identify their determinants. Six items were removed from the item analysis. EFA supported the existence of two factors. CFA results indicated that the two-factor model fit the data better than the one-factor model. Logistic regression analyses indicated that being female, majoring in non-health-related disciplines, attributing homosexuality to uncontrollable causes, non-adherence to traditional gender norms and exposure to homosexual content were significantly associated with less negative attitudes toward both L and G. Urban students were marginally less likely to express negative attitudes toward L but not G, while non-heterosexuals and those who had prior personal contact with homosexuals exhibited less negative attitudes toward G but not L. However, grade showed no significant associations with either ATL or ATG. The retained 14-item version of Herek’s ATLG scale has been proven to be a reliable and valid tool. Furthermore, ATL and ATG were determined by different factors and thus would be treated separately. In order to reduce negative attitudes toward LG among undergraduates in mainland China, a comprehensive intervention plan such as conducting comprehensive sex education and pushing the process of legalizing same-sex marriage should be designed, implemented and evaluated. (shrink)