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<b>Background/Objectives:</b> 3′-Sialyllactose (3′-SL), a human milk oligosaccharide, has anti-inflammatory effects and is demonstrated to have protective effects against osteoarthritis (OA) in vitro and in vivo. However, this hypothesis remains to be investigated in a clinical setting. Herein, we investigated the effects of 3′-SL on pain and physical function in patients with knee OA. <b>Methods:</b> Sixty patients with knee OA with Kellgren and Lawrence grades (KL-grades) 1–4 and Korean Western Ontario and McMaster Universities Osteoarthritis Index (KWOMAC) scores ≥30 were randomly assigned to the placebo (<i>n</i> = 20), 3′-SL 200 mg (<i>n</i> = 20), and 3′-SL 600 mg (<i>n</i> = 20) groups. For 12 weeks, 3′-SL or placebo was administered to patients once a day. Clinical efficacy was evaluated using a visual analog scale (VAS) for pain and KWOMAC for physical function at baseline and at 6 and 12 weeks. Adverse effects were assessed for 12 weeks. <b>Results:</b> Significant reductions in VAS and KWOMAC scores were observed at 12 weeks compared with the baseline in the 3′-SL group. No severe adverse effects were observed over 12 weeks. <b>Conclusions:</b> 3′-SL reduced pain in patients with knee OA, improved daily life movements, and was safe, suggesting that 3′-SL might be an effective treatment for knee OA without severe side effects.