Nursing assistants (NAs) working in nursing homes (NHs) are at higher risk for work-related musculoskeletal symptoms (WRMSs) than their counterparts working in other health care settings. Worldwide, NAs have ranked shoulders in the top three body parts at risk of WRMSs. However, factors associated with their shoulder WRMSs are currently unknown. The aim of this study was to identify these associated risk factors among NAs working in NHs. 440 NAs from 47 nursing homes (with 60–90% response rate from each nursing home), recruited by convenience sampling, participated in this cross-sectional study in 2014–2015. A validated and reliable questionnaire was used for data collection. Information on demographic, job content questionnaire (JCQ), perceived physical exertion (PE), workstyle, ergonomic and manual handling knowledge and other work-related factors was collected using a self-administered questionnaire. 53% of the participants reported experiencing with WRMSs in their shoulders. Nine associated factors of shoulder WRMSs were identified using bivariate analysis. With the adjustment of age and gender using multivariable logistic regression, body mass index (OR = .931, 95% CI [.874–.991]), job title of health workers (OR = 2.72, 95% CI [1.18–6.25]) and workstyle-working through pain (OR = 1.06, 95% CI [1.01–1.11]) remained as predictors. Effort should be directed at integrating “workstyle intervention” into lifestyle physical activity training for NAs.