Contraceptive options for men are limited to either condom use or surgical vasectomy. Ongoing scientific efforts seek to expand existing male contraceptive options to include reversible options with high efficacy and reliability. Herein, we formulated EP055, a novel non-hormonal compound with reversible contraceptive effect, into an in-situ forming implant (ISFI) to demonstrate potential of male contraception with a long-acting injectable. Over a dozen ISFI formulations were studied, though release durations were limited due to the hydrophilic nature of EP055. An optimized EP055-ISFI formulation (F.04) elicited sustained release in vitro over 35 days and was further investigated in vivo for safety, pharmacokinetics (PK), and efficacy in male BALB/c mice. Plasma EP055 concentrations elicited high burst release in the first 24 h followed by first order-like release kinetics up to day 14 and sustained release between day 14-28. EP055 ISFI removal resulted in a rapid decline of EP055 plasma concentration, which fell below the limit of quantification. A reduction in sperm motility and an increase in premature acrosomal membrane degradation were observed with sperm samples collected at day 3 post EP055-ISFI administration, indicating contraceptive efficacy. Furthermore, EP055 was well-tolerated with no signs of systemic inflammation. Collectively, these results support future development of EPPIN-targeting molecules and in-situ forming implants for male contraception.