Background: Due to declining birthrates and an increasing aging population, shortage of the caregiving labor force has become a global issue. Among various efforts toward the solution, introducing robotic products for assistance could provide an effective way to help older adults in their daily lives. As previous studies have indicated that older adults’ acceptance of robots is lower than that of younger adults, enhancing older adults’ acceptance of robots is imperative. Because older adults’ first impressions based on a robot’s appearance might affect their acceptance of the robot, we investigated the uncanny valley effect (UVE) here. The UVE refers to the phenomenon that people rate robots more positively as robots become more humanlike, but only up to a certain point; as robots approach a near-perfect similarity to human appearance, likeability drops and forms the uncanny valley. Nevertheless, evidence for the UVE came mainly from younger adults. Objective: The present study aimed to examine whether the UVE varies across different age groups and whether a robot’s appearance would affect participants’ acceptance of the robot’s service or companionship. Methods: An online questionnaire study was conducted with 255 participants, including younger (n = 77, age 18–39 years), middle-aged (n = 87, age 40–59 years), and older (n = 91, age 60–87 years) adults. Participants were asked to view each picture in a set selected from a total of 83 robot pictures and evaluate their impressions of each robot and the intention of use regarding robot function as a service provider or a companion. Results: The UVE was found in younger and middle-aged adults; however, older adults did not show the UVE. Older adults preferred humanlike over non-humanlike robots, regardless of robot function. Conclusion: The design of assistive robots should take the UVE into consideration by customizing robot appearance based on the age group of the intended user.