The purpose of this study was to develop and test a research model that investigates the factors influencing knowledge acquisition among adult workers participating in online informal learning activities. A total of 342 adult workers, all of whom were civil servants working for the government in Taiwan, participated in this study. A survey instrument was used to assess their perceived abilities in information querying, information differentiation, online communication self-efficacy, information evaluation and extraction, and knowledge acquisition. A Structural Equation Modeling was performed using Partial Least Squares regression for data exploration and model estimation. The results of this study indicate that information querying, significantly affected the participants’ information differentiation performance. The construct of information differentiation yielded a positive influence in the respondents’ online communication self-efficacy and information evaluation and extraction. Moreover, information evaluation and extraction was found to be a significant predictor influencing adult workers’ knowledge acquisition. The findings imply that organizations should consider including information literacy training in training initiatives and provide friendly online collaborating tools to enhance employees’ online informal learning experiences.