Aim To investigate cases with conjunctival pigmentation originating from sclerotomy sites after 23-gauge microincisional vitrectomy surgery (MIVS). Methods This is a retrospective study comprised of 185 eyes from 185 consecutive patients who underwent 23-gauge MIVS. The authors investigated the incidence and the clinical factors associated with conjunctival pigmentation, including indication of vitrectomy and surgical procedures. Results Eight eyes (4.3%) from 185 eyes showed conjunctival pigmentation after surgery within 7 days postoperatively. Light microscopic examination of excised tissue showed infiltration of abundant dark brownish colored melanophages with surrounding scattered, finely granular, melanin pigments. C3F8 gas tamponade (OR, 9.4; 95% CI, 1.9-44.5; P ¼ 0.005) was significantly associated with conjunctival pigmentation. The mean surface area (21.26 ± 2.21 mm 2 ) of three eyes with C3F8 tamponade was significantly larger than that of five eyes (2.51 ± 0.73 mm 2 ) without tamponade (P ¼ 0.025). Neither leakage nor hypotony was found. Conclusions Intraocular melanin pigments could prolapse through the sclerotomy site and cause conjunctival pigmentation after 23-gauge MIVS. Thus, preoperative warning is necessary in patients with 23-gauge MIVS.