Background: This study evaluated the effects of a 3-month workplace physical activity (PA) intervention on employees’ health-related fitness and well-being. Moreover, mediational pathways were examined. Methods: A longitudinal, quasi-experimental trial was evaluated in Belgian employees. The intervention group (IG: n = 246) received 3-months individualized, tailored PA counseling, consisting of face-to-face counseling and follow-up e-mail and telephone contacts. The reference group (n = 54) received no PA counseling. Outcome measures (assessed at baseline, 3 mo [short term], and 9 mo [long term]) included body composition, cardiorespiratory fitness, well-being, and step-based PA. Results: With no changes in the reference group, the IG showed short-term improvements in body mass index (−0.24 kg/m2, P < .001), body fat (−0.88%, P < .001), waist circumference (−1.47 cm, P < .001), and muscle percentage (+0.47%, P < .001). Moreover, with respect to cardiorespiratory fitness, IG participants improved on perceived exertion, both in the short term (−0.91, P < .001) and long term (−0.83, P < .001). Furthermore, perceived physical well-being increased in the IG, both in the short term (+0.57, P < .001) and long term (+0.57, P < .001). The observed intervention effects on body composition, perceived exertion, and well-being were mediated by (increases in) step-based PA. Conclusions: Workplace PA counseling programs have the potential to enhance employees’ health-related fitness and well-being. Importantly, step-based PA behavior change was found to contribute to the postintervention improvements.