This study provided knowledge on testicular development, spawning reference and length at first maturity (Lm) of Acentrogobius viridipunctatus—a target catching fish for food supply in the southwest Viet Nam. A total of 528 individuals were collected using trawl nets from January to December 2020 at Long Huu-Tra Vinh (LHTV), Trung Binh-Soc Trang (TBST), Dien Hai-Bac Lieu (DHBL) and Tan Thuan-Ca Mau (TTCM). Five developmental stages of testis were found, and testis diameter increased from ~1.07 (stage I) to ~4.82 mm (stage V). Testis stage I consisted of spermatogonia (S) which shifted to primary spermatocytes (SC1) in stage II. In testis stage III, SC1 was divided into secondary spermatocytes (SC2). At this stage, testis consisted of S, SC1, SC2 and some spermatid (ST). At stages IV and V, testis comprised mainly spermatozoa (SZ) besides S, SC1, SC2 and ST, showing that this goby was a multi-spawner. Testis frequency and gonadosomatic index analyses showed that this species released sperms yearly, with a prominent peak in the wet season. The Lm decreased from 9.3  0.3 cm at TBST to 9.0  0.3 cm at LHTV, 8.9  0.2 cm at TTCM and 6.5  0.5 cm at DHBL. Findings provided fundamental data to set up suitable fishing and conserving strategies for this species.