The current study evaluates guessing behaviors in a vocabulary size test (VST) and examines whether including an "I don't know" in a VST may have an impact on the results of the test. One-hundred-fifty first-year students at a university in China took part in the study. They were randomly assigned to three groups. Each group took a different version of the VST: the original VST, the VST with an "I don't know" option, and the VST with an "I don't know" option and a penalty instruction. After the VST, a reading comprehension test (used as a distraction) and a meaning recall task were administered to all participants. Comparing the results of the VST and the recall task, it was found that guessing behaviors can be influenced by factors such as vocabulary frequency level, partial knowledge, and the multiple-choice options. Results also indicate that the "I don't know option" not only reduced the number of guesses but also discouraged partial knowledge. Whether to include the "I don't know option" and the penalty instruction depends on how the test is to be used.